Academic Appointments

Administrative Appointments

  • Faculty Lead, Stanford University School of Medicine Propel Postdoctoral Scholars Program (2022 - Present)
  • Director of Research Operations, Department of Radiology, Stanford University (2020 - Present)

Honors & Awards

  • Distinguished Investigator Award, The Academy for Radiology and Biomedical Imaging Research (2021)
  • Breast Cancer Research Program Breakthrough Award, Department of Defense - Congressionally Directed Medical Research Programs (2015)
  • Innovative Development and Exploratory Award, California Breast Cancer Research Program (2013)
  • Research Award, American Society for Mass Spectrometry (2012)
  • McCormick Faculty Award, Stanford University School of Medicine (2012)
  • Developmental Cancer Research Award, Stanford Cancer Institute (2011, 2013)

Boards, Advisory Committees, Professional Organizations

  • Treasurer, American Society of Mass Spectrometry Board of Directors (2022 - Present)
  • Vice Chair, California Breast Cancer Research Council (2022 - Present)
  • Member, California Breast Cancer Research Council (2019 - Present)
  • Scientific Advisory Board Member, OHSU Knight Cancer Institute Cancer Early Detection Advanced Research Center (CEDAR) (2019 - Present)

Professional Education

  • Postdoctoral Research, Fred Hutchinson Cancer Research Center (2010)
  • PhD, Purdue University, Chemistry (2005)
  • BA, Carleton College, Chemistry (2001)

Current Research and Scholarly Interests

The Pitteri laboratory is focused on the discovery and validation of proteins that can be used as molecular indicators of risk, diagnosis, progression, and recurrence of cancer. Proteomic technologies, predominantly mass spectrometry, are used to identify proteins in the blood that are differentially regulated and/or post-translationally modified with disease state. Using human plasma samples, tumor tissue, cancer cell lines, and genetically engineered mouse models, the origins of these proteins are being investigated. A major goal of this research is to define novel molecular signatures for breast and ovarian cancers, including particular sub-types of these diseases. This laboratory is also focused on the identification of proteins with expression restricted to the surface of cancer cells which can be used as novel targets for molecular imaging technologies.

Stanford Advisees

  • Doctoral Dissertation Reader (AC)
    Tim Chai
  • Postdoctoral Faculty Sponsor
    Katie Kapp
  • Doctoral Dissertation Advisor (AC)
    Claudia Leonard

Graduate and Fellowship Programs

All Publications

  • Inhibition of protein translational machinery in triple-negative breast cancer as a promising therapeutic strategy. Cell reports. Medicine Dheeraj, A., Garcia Marques, F. J., Tailor, D., Bermudez, A., Resendez, A., Pandrala, M., Grau, B., Kumar, P., Haley, C. B., Honkala, A., Kujur, P., Jeffrey, S. S., Pitteri, S., Malhotra, S. V. 2024: 101552


    Y-box binding protein-1 (YB-1) is a proto-oncogenic protein associated with protein translation regulation. It plays a crucial role in the development and progression of triple-negative breast cancer (TNBC). In this study, we describe a promising approach to inhibit YB-1 using SU056, a small-molecule inhibitor. SU056 physically interacts with YB-1 and reduces its expression, which helps to restrain the progression of TNBC. Proteome profiling analysis indicates that the inhibition of YB-1 by SU056 can alter the proteins that regulate protein translation, an essential process for cancer cell growth. Preclinical studies on human cells, mice, and patient-derived xenograft tumor models show the effectiveness of SU056. Moreover, toxicological studies have shown that SU056 treatment and dosing are well tolerated without any adverse effects. Overall, our study provides a strong foundation for the further development of SU056 as a potential treatment option for patients with TNBC by targeting YB-1.

    View details for DOI 10.1016/j.xcrm.2024.101552

    View details for PubMedID 38729158

  • AZGP1 deficiency promotes angiogenesis in prostate cancer. Journal of translational medicine Wen, R. M., Qiu, Z., Marti, G. E., Peterson, E. E., Marques, F. J., Bermudez, A., Wei, Y., Nolley, R., Lam, N., Polasko, A. L., Chiu, C. L., Zhang, D., Cho, S., Karageorgos, G. M., McDonough, E., Chadwick, C., Ginty, F., Jung, K. J., Machiraju, R., Mallick, P., Crowley, L., Pollack, J. R., Zhao, H., Pitteri, S. J., Brooks, J. D. 2024; 22 (1): 383


    Loss of AZGP1 expression is a biomarker associated with progression to castration resistance, development of metastasis, and poor disease-specific survival in prostate cancer. However, high expression of AZGP1 cells in prostate cancer has been reported to increase proliferation and invasion. The exact role of AZGP1 in prostate cancer progression remains elusive.AZGP1 knockout and overexpressing prostate cancer cells were generated using a lentiviral system. The effects of AZGP1 under- or over-expression in prostate cancer cells were evaluated by in vitro cell proliferation, migration, and invasion assays. Heterozygous AZGP1± mice were obtained from European Mouse Mutant Archive (EMMA), and prostate tissues from homozygous knockout male mice were collected at 2, 6 and 10 months for histological analysis. In vivo xenografts generated from AZGP1 under- or over-expressing prostate cancer cells were used to determine the role of AZGP1 in prostate cancer tumor growth, and subsequent proteomics analysis was conducted to elucidate the mechanisms of AZGP1 action in prostate cancer progression. AZGP1 expression and microvessel density were measured in human prostate cancer samples on a tissue microarray of 215 independent patient samples.Neither the knockout nor overexpression of AZGP1 exhibited significant effects on prostate cancer cell proliferation, clonal growth, migration, or invasion in vitro. The prostates of AZGP1-/- mice initially appeared to have grossly normal morphology; however, we observed fibrosis in the periglandular stroma and higher blood vessel density in the mouse prostate by 6 months. In PC3 and DU145 mouse xenografts, over-expression of AZGP1 did not affect tumor growth. Instead, these tumors displayed decreased microvessel density compared to xenografts derived from PC3 and DU145 control cells, suggesting that AZGP1 functions to inhibit angiogenesis in prostate cancer. Proteomics profiling further indicated that, compared to control xenografts, AZGP1 overexpressing PC3 xenografts are enriched with angiogenesis pathway proteins, including YWHAZ, EPHA2, SERPINE1, and PDCD6, MMP9, GPX1, HSPB1, COL18A1, RNH1, and ANXA1. In vitro functional studies show that AZGP1 inhibits human umbilical vein endothelial cell proliferation, migration, tubular formation and branching. Additionally, tumor microarray analysis shows that AZGP1 expression is negatively correlated with blood vessel density in human prostate cancer tissues.AZGP1 is a negative regulator of angiogenesis, such that loss of AZGP1 promotes angiogenesis in prostate cancer. AZGP1 likely exerts heterotypical effects on cells in the tumor microenvironment, such as stromal and endothelial cells. This study sheds light on the anti-angiogenic characteristics of AZGP1 in the prostate and provides a rationale to target AZGP1 to inhibit prostate cancer progression.

    View details for DOI 10.1186/s12967-024-05183-x

    View details for PubMedID 38659028

    View details for PubMedCentralID 321763

  • Identification and characterization of intact glycopeptides in human urine. Scientific reports Garcia-Marques, F., Fuller, K., Bermudez, A., Shamsher, N., Zhao, H., Brooks, J. D., Flory, M. R., Pitteri, S. J. 2024; 14 (1): 3716


    Glycoproteins in urine have the potential to provide a rich class of informative molecules for studying human health and disease. Despite this promise, the urine glycoproteome has been largely uncharacterized. Here, we present the analysis of glycoproteins in human urine using LC-MS/MS-based intact glycopeptide analysis, providing both the identification of protein glycosites and characterization of the glycan composition at specific glycosites. Gene enrichment analysis reveals differences in biological processes, cellular components, and molecular functions in the urine glycoproteome versus the urine proteome, as well as differences based on the major glycan class observed on proteins. Meta-heterogeneity of glycosylation is examined on proteins to determine the variation in glycosylation across multiple sites of a given protein with specific examples of individual sites differing from the glycosylation trends in the overall protein. Taken together, this dataset represents a potentially valuable resource as a baseline characterization of glycoproteins in human urine for future urine glycoproteomics studies.

    View details for DOI 10.1038/s41598-024-53299-3

    View details for PubMedID 38355753

    View details for PubMedCentralID PMC10866872

  • UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas. Cell reports. Medicine Liu, S., Chai, T., Garcia-Marques, F., Yin, Q., Hsu, E. C., Shen, M., Shaw Toland, A. M., Bermudez, A., Hartono, A. B., Massey, C. F., Lee, C. S., Zheng, L., Baron, M., Denning, C. J., Aslan, M., Nguyen, H. M., Nolley, R., Zoubeidi, A., Das, M., Kunder, C. A., Howitt, B. E., Soh, H. T., Weissman, I. L., Liss, M. A., Chin, A. I., Brooks, J. D., Corey, E., Pitteri, S. J., Huang, J., Stoyanova, T. 2024: 101381


    Neuroendocrine carcinomas, such as neuroendocrine prostate cancer and small-cell lung cancer, commonly have a poor prognosis and limited therapeutic options. We report that ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is elevated in tissues and plasma from patients with neuroendocrine carcinomas. Loss of UCHL1 decreases tumor growth and inhibits metastasis of these malignancies. UCHL1 maintains neuroendocrine differentiation and promotes cancer progression by regulating nucleoporin, POM121, and p53. UCHL1 binds, deubiquitinates, and stabilizes POM121 to regulate POM121-associated nuclear transport of E2F1 and c-MYC. Treatment with the UCHL1 inhibitor LDN-57444 slows tumor growth and metastasis across neuroendocrine carcinomas. The combination of UCHL1 inhibitors with cisplatin, the standard of care used for neuroendocrine carcinomas, significantly delays tumor growth in pre-clinical settings. Our study reveals mechanisms of UCHL1 function in regulating the progression of neuroendocrine carcinomas and identifies UCHL1 as a therapeutic target and potential molecular indicator for diagnosing and monitoring treatment responses in these malignancies.

    View details for DOI 10.1016/j.xcrm.2023.101381

    View details for PubMedID 38244540

  • Developing and Applying Intact Glycoproteomics Analysis Workflows for Cancer Pitteri, S. OXFORD UNIV PRESS INC. 2023: 1068
  • Guanylate-binding protein 1 modulates proteasomal machinery in ovarian cancer. iScience Tailor, D., Garcia-Marques, F. J., Bermudez, A., Pitteri, S. J., Malhotra, S. V. 2023; 26 (11): 108292


    Guanylate-binding protein 1 (GBP1) is known as an interferon-γ-induced GTPase. Here, we used genetically modified ovarian cancer (OC) cells to study the role of GBP1. The data generated show that GBP1 inhibition constrains the clonogenic potential of cancer cells. In vivo studies revealed that GBP1 overexpression in tumors promotes tumor progression and reduces median survival, whereas GBP1 inhibition delayed tumor progression with longer median survival. We employed proteomics-based thermal stability assay (CETSA) on GBP1 knockdown and overexpressed OC cells to study its molecular functions. CETSA results show that GBP1 interacts with many members of the proteasome. Furthermore, GBP1 inhibition sensitizes OC cells to paclitaxel treatment via accumulated ubiquitinylated proteins where GBP1 inhibition decreases the overall proteasomal activity. In contrast, GBP1-overexpressing cells acquired paclitaxel resistance via boosted cellular proteasomal activity. Overall, these studies expand the role of GBP1 in the activation of proteasomal machinery to acquire chemoresistance.

    View details for DOI 10.1016/j.isci.2023.108292

    View details for PubMedID 38026225

    View details for PubMedCentralID PMC10665831

  • A drug-like molecule engages nuclear hormone receptor DAF-12/FXR to regulate mitophagy and extend lifespan. Nature aging Chamoli, M., Rane, A., Foulger, A., Chinta, S. J., Shahmirzadi, A. A., Kumsta, C., Nambiar, D. K., Hall, D., Holcom, A., Angeli, S., Schmidt, M., Pitteri, S., Hansen, M., Lithgow, G. J., Andersen, J. K. 2023


    Autophagy-lysosomal function is crucial for maintaining healthy lifespan and preventing age-related diseases. The transcription factor TFEB plays a key role in regulating this pathway. Decreased TFEB expression is associated with various age-related disorders, making it a promising therapeutic target. In this study, we screened a natural product library and discovered mitophagy-inducing coumarin (MIC), a benzocoumarin compound that enhances TFEB expression and lysosomal function. MIC robustly increases the lifespan of Caenorhabditis elegans in an HLH-30/TFEB-dependent and mitophagy-dependent manner involving DCT-1/BNIP3 while also preventing mitochondrial dysfunction in mammalian cells. Mechanistically, MIC acts by inhibiting ligand-induced activation of the nuclear hormone receptor DAF-12/FXR, which, in turn, induces mitophagy and extends lifespan. In conclusion, our study uncovers MIC as a promising drug-like molecule that enhances mitochondrial function and extends lifespan by targeting DAF-12/FXR. Furthermore, we discovered DAF-12/FXR as a previously unknown upstream regulator of HLH-30/TFEB and mitophagy.

    View details for DOI 10.1038/s43587-023-00524-9

    View details for PubMedID 37957360

    View details for PubMedCentralID 8659158

  • The role of GCNT1 mediated O-glycosylation in aggressive prostate cancer. Scientific reports Hodgson, K., Orozco-Moreno, M., Scott, E., Garnham, R., Livermore, K., Thomas, H., Zhou, Y., He, J., Bermudez, A., Garcia Marques, F. J., Bastian, K., Hysenaj, G., Archer Goode, E., Heer, R., Pitteri, S., Wang, N., Elliott, D. J., Munkley, J. 2023; 13 (1): 17031


    Prostate cancer is the most common cancer in men and a major cause of cancer related deaths worldwide. Nearly all affected men develop resistance to current therapies and there is an urgent need to develop new treatments for advanced disease. Aberrant glycosylation is a common feature of cancer cells implicated in all of the hallmarks of cancer. A major driver of aberrant glycosylation in cancer is the altered expression of glycosylation enzymes. Here, we show that GCNT1, an enzyme that plays an essential role in the formation of core 2 branched O-glycans and is crucial to the final definition of O-glycan structure, is upregulated in aggressive prostate cancer. Using in vitro and in vivo models, we show GCNT1 promotes the growth of prostate tumours and can modify the glycome of prostate cancer cells, including upregulation of core 2 O-glycans and modifying the O-glycosylation of secreted glycoproteins. Furthermore, using RNA sequencing, we find upregulation of GCNT1 in prostate cancer cells can alter oncogenic gene expression pathways important in tumour growth and metastasis. Our study highlights the important role of aberrant O-glycosylation in prostate cancer progression and provides novel insights regarding the mechanisms involved.

    View details for DOI 10.1038/s41598-023-43019-8

    View details for PubMedID 37813880

    View details for PubMedCentralID 8945091

  • Measuring the multifaceted roles of mucin-domain glycoproteins in cancer. Advances in cancer research Riley, N. M., Wen, R. M., Bertozzi, C. R., Brooks, J. D., Pitteri, S. J. 2023; 157: 83-121


    Mucin-domain glycoproteins are highly O-glycosylated cell surface and secreted proteins that serve as both biochemical and biophysical modulators. Aberrant expression and glycosylation of mucins are known hallmarks in numerous malignancies, yet mucin-domain glycoproteins remain enigmatic in the broad landscape of cancer glycobiology. Here we review the multifaceted roles of mucins in cancer through the lens of the analytical and biochemical methods used to study them. We also describe a collection of emerging tools that are specifically equipped to characterize mucin-domain glycoproteins in complex biological backgrounds. These approaches are poised to further elucidate how mucin biology can be understood and subsequently targeted for the next generation of cancer therapeutics.

    View details for DOI 10.1016/bs.acr.2022.09.001

    View details for PubMedID 36725114

  • ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression. The Journal of pathology Scott, E., Archer Goode, E., Garnham, R., Hodgson, K., Orozco-Moreno, M., Turner, H., Livermore, K., Putri Nangkana, K., Frame, F. M., Bermudez, A., Jose Garcia Marques, F., McClurg, U. L., Wilson, L., Thomas, H., Buskin, A., Hepburn, A., Duxfield, A., Bastian, K., Pye, H., Arredondo, H. M., Hysenaj, G., Heavey, S., Stopka-Farooqui, U., Haider, A., Freeman, A., Singh, S., Johnston, E. W., Punwani, S., Knight, B., McCullagh, P., McGrath, J., Crundwell, M., Harries, L., Heer, R., Maitland, N. J., Whitaker, H., Pitteri, S., Troyer, D. A., Wang, N., Elliott, D. J., Drake, R. R., Munkley, J. 2023


    Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in alpha2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched alpha2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces alpha2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find alpha2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and alpha2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

    View details for DOI 10.1002/path.6152

    View details for PubMedID 37550801

  • Lineage plasticity in SCLC generates non-neuroendocrine cells primed for vasculogenic mimicry. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer Pearsall, S. M., Williamson, S. C., Humphrey, S., Hughes, E., Morgan, D., García Marqués, F. J., Awanis, G., Carroll, R., Burks, L., Shue, Y. T., Bermudez, A., Frese, K. K., Galvin, M., Carter, M., Priest, L., Kerr, A., Zhou, C., Oliver, T. G., Humphries, J. D., Humphries, M. J., Blackhall, F., Cannell, I. G., Pitteri, S. J., Hannon, G. J., Sage, J., Dive, C., Simpson, K. L. 2023


    Vasculogenic mimicry (VM), the process of tumor cell trans-differentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including small cell lung cancer (SCLC). In genetically engineered mouse models (GEMMs) of SCLC, NOTCH and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs.We analysed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs and patient biopsies. VM-proficient cell subpopulations in ex vivo cultures were molecularly profiled by RNA sequencing and mass spectrometry. We evaluated their 3D structure and defined collagen-integrin interactions.We show that VM vessels are present in 23/25 CDX models, 2 GEMMs and in 20 patient biopsies of SCLC. Perfused VM vessels support tumor growth and only Notch-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development and extracellular matrix (ECM) organization signatures. On Matrigel, VM-primed non-NE cells re-model ECM into hollow tubules in an integrin β1-dependent process.We identify VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take significant steps towards understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve SCLC patient outcomes in the future.

    View details for DOI 10.1016/j.jtho.2023.07.012

    View details for PubMedID 37455012

  • Proteomics analysis of urine and catheter-associated biofilms in spinal cord injury patients. American journal of clinical and experimental urology Garcia-Marques, F. J., Zakrasek, E., Bermudez, A., Polasko, A. L., Liu, S., Stoyanova, T., Brooks, J. D., Lavelle, J., Pitteri, S. J. 2023; 11 (3): 206-219


    After spinal cord injury (SCI), use chronic urinary catheters for bladder management is common, making these patients especially vulnerable to catheter-associated complications. Chronic catheterization is associated with bacterial colonization and frequent catheter-associated urinary tract infections (CAUTI). One determinant of infection success and treatment resistance is production of catheter-associated biofilms, composed of microorganisms and host- and microbial-derived components. To better understand the biofilm microenvironment, we performed proteomics analysis of catheter-associated biofilms and paired urine samples from four people with SCI with chronic indwelling urinary catheters. We developed a novel method for the removal of adhered cellular components on catheters that contained both human and microbial homologous proteins. Proteins from seven microbial species were identified including: Escherichia coli, Klebsiella species (spp), Enterococcus spp, Proteus mirabilis, Pseudomonas spp, Staphylococcus spp, and Candida spp. Peptides identified from catheter biofilms were assigned to 4,820 unique proteins, with 61% of proteins assigned to the biofilm-associated microorganisms, while the remainder were human-derived. Contrastingly, in urine, only 51% were assigned to biofilm-associated microorganisms and 4,554 proteins were identified as a human-derived. Of the proteins assigned to microorganisms in the biofilm and paired urine, Enterococcus, Candida spp, and P. mirabilis had greater associations with the biofilm phase, whereas E. coli and Klebsiella had greater associations with the urine phase, thus demonstrating a significant difference between the urine and adhered microbial communities. The microbial proteins that differed significantly between the biofilm and paired urine samples mapped to pathways associated with amino acid synthesis, likely related to adaptation to high urea concentrations in the urine, and growth and protein synthesis in bacteria in the biofilm. Human proteins demonstrated enrichment for immune response in the catheter-associated biofilm. Proteomic analysis of catheter-associated biofilms and paired urine samples has the potential to provide detailed information on host and bacterial responses to chronic indwelling urinary catheters and could be useful for understanding complications of chronic indwelling catheters including CAUTIs, urinary stones, and catheter blockages.

    View details for PubMedID 37441441

    View details for PubMedCentralID PMC10333135

  • Siglec-7/9 are novel immune checkpoints for prostate cancer Wen, R., Stark, J. C., Marti, G., Garcia-Marques, F., Zhao, H., Nolley, R., Bertozzi, C. R., Pitteri, S. J., Brooks, J. D. AMER ASSOC IMMUNOLOGISTS. 2023
  • Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth. Oncogene Scott, E., Hodgson, K., Calle, B., Turner, H., Cheung, K., Bermudez, A., Marques, F. J., Pye, H., Yo, E. C., Islam, K., Oo, H. Z., McClurg, U. L., Wilson, L., Thomas, H., Frame, F. M., Orozco-Moreno, M., Bastian, K., Arredondo, H. M., Roustan, C., Gray, M. A., Kelly, L., Tolson, A., Mellor, E., Hysenaj, G., Goode, E. A., Garnham, R., Duxfield, A., Heavey, S., Stopka-Farooqui, U., Haider, A., Freeman, A., Singh, S., Johnston, E. W., Punwani, S., Knight, B., McCullagh, P., McGrath, J., Crundwell, M., Harries, L., Bogdan, D., Westaby, D., Fowler, G., Flohr, P., Yuan, W., Sharp, A., de Bono, J., Maitland, N. J., Wisnovsky, S., Bertozzi, C. R., Heer, R., Guerrero, R. H., Daugaard, M., Leivo, J., Whitaker, H., Pitteri, S., Wang, N., Elliott, D. J., Schumann, B., Munkley, J. 2023


    Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression.

    View details for DOI 10.1038/s41388-023-02604-x

    View details for PubMedID 36725887

  • Integrated transcriptome-proteome analyses of human stem cells reveal source-dependent differences in their regenerative signature. Stem cell reports Ganguly, A., Swaminathan, G., Garcia-Marques, F., Regmi, S., Yarani, R., Primavera, R., Chetty, S., Bermudez, A., Pitteri, S. J., Thakor, A. S. 2022


    Mesenchymal stem cells (MSCs) are gaining increasing prominence as an effective regenerative cellular therapy. However, ensuring consistent and reliable effects across clinical populations has proved to be challenging. In part, this can be attributed to heterogeneity in the intrinsic molecular and regenerative signature of MSCs, which is dependent on their source of origin. The present work uses integrated omics-based profiling, at different functional levels, to compare the anti-inflammatory, immunomodulatory, and angiogenic properties between MSCs from neonatal (umbilical cord MSC [UC-MSC]) and adult (adipose tissue MSC [AD-MSC], and bone marrow MSC [BM-MSC]) sources. Using multi-parametric analyses, we identified that UC-MSCs promote a more robust host innate immune response; in contrast, adult-MSCs appear to facilitate remodeling of the extracellular matrix (ECM) with stronger activation of angiogenic cascades. These data should help facilitate the standardization of source-specific MSCs, such that their regenerative signatures can be confidently used to target specific disease processes.

    View details for DOI 10.1016/j.stemcr.2022.11.006

    View details for PubMedID 36493779

  • Siglec-7/9-sialic acid interactions inhibit T cell immune response in prostate cancer Wen, R. M., Stark, J., Garcia-Marques, F., Nolley, H., Bertozzi, C. R., Pitteri, S. J., Brooks, J. D. AMER ASSOC CANCER RESEARCH. 2022
  • Inhibiting ribosomal proteins with a small molecule: Therapeutic strategy for triple negative breast cancer Dheeraj, A., Tailor, D., Resendez, A., Marques, F., Bermudez, A., Pitteri, S., Malhotra, S. AMER ASSOC CANCER RESEARCH. 2022
  • Engineering genetically-encoded synthetic biomarkers for breath-based cancer detection Vermesh, O., D'Souza, A., Alam, I., Wardak, M., McLaughlin, T., El Rami, F., Sathirachinda, A., Bell, J., Pitteri, S., James, M., Hori, S., Gross, E., Gambhir, S. AMER ASSOC CANCER RESEARCH. 2022
  • Inhibition of triple negative breast cancer metastasis via Enolase-1 modulation Tailor, D., Dheeraj, A., Garcia-Marques, F., Pandrala, M., Bermudez, A., Pitteri, S., Malhotra, S. V. AMER ASSOC CANCER RESEARCH. 2022
  • SU086, an inhibitor of HSP90, impairs glycolysis and represents a treatment strategy for advanced prostate cancer. Cell reports. Medicine Rice, M. A., Kumar, V., Tailor, D., Garcia-Marques, F. J., Hsu, E., Liu, S., Bermudez, A., Kanchustambham, V., Shankar, V., Inde, Z., Alabi, B. R., Muruganantham, A., Shen, M., Pandrala, M., Nolley, R., Aslan, M., Ghoochani, A., Agarwal, A., Buckup, M., Kumar, M., Going, C. C., Peehl, D. M., Dixon, S. J., Zare, R. N., Brooks, J. D., Pitteri, S. J., Malhotra, S. V., Stoyanova, T. 2022; 3 (2): 100502


    Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells invitro, cell-line and patient-derived xenografts invivo, and exvivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth invitro and invivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.

    View details for DOI 10.1016/j.xcrm.2021.100502

    View details for PubMedID 35243415

  • Protein signatures to distinguish aggressive from indolent prostate cancer. The Prostate Garcia-Marques, F., Liu, S., Totten, S. M., Bermudez, A., Tanimoto, C., Hsu, E. C., Nolley, R., Hembree, A., Stoyanova, T., Brooks, J. D., Pitteri, S. J. 2022


    Distinguishing men with aggressive from indolent prostate cancer is critical to decisions in the management of clinically localized prostate cancer. Molecular signatures of aggressive disease could help men overcome this major clinical challenge by reducing unnecessary treatment and allowing more appropriate treatment of aggressive disease.We performed a mass spectrometry-based proteomic analysis of normal and malignant prostate tissues from 22 men who underwent surgery for prostate cancer. Prostate cancer samples included Grade Groups (3-5), with 8 patients experiencing recurrence and 14 without evidence of recurrence with a mean of 6.8 years of follow-up. To better understand the biological pathways underlying prostate cancer aggressiveness, we performed a systems biology analysis and gene enrichment analysis. Proteins that distinguished recurrent from nonrecurrent cancer were chosen for validation by immunohistochemical analysis on tissue microarrays containing samples from a larger cohort of patients with recurrent and nonrecurrent prostate cancer.In all, 24,037 unique peptides (false discovery rate < 1%) corresponding to 3,313 distinct proteins were identified with absolute abundance ranges spanning seven orders of magnitude. Of these proteins, 115 showed significantly (p < 0.01) different levels in tissues from recurrent versus nonrecurrent cancers. Analysis of all differentially expressed proteins in recurrent and nonrecurrent cases identified several protein networks, most prominently one in which approximately 24% of the proteins in the network were regulated by the YY1 transcription factor (adjusted p < 0.001). Strong immunohistochemical staining levels of three differentially expressed proteins, POSTN, CALR, and CTSD, on a tissue microarray validated their association with shorter patient survival.The protein signatures identified could improve understanding of the molecular drivers of aggressive prostate cancer and be used as candidate prognostic biomarkers.

    View details for DOI 10.1002/pros.24307

    View details for PubMedID 35098564

  • Engineered Cell-Derived Vesicles Displaying Targeting Peptide and Functionalized with Nanocarriers for Therapeutic microRNA Delivery to Triple-Negative Breast Cancer in Mice. Advanced healthcare materials Bose, R. J., Kumar, U. S., Garcia-Marques, F., Zeng, Y., Habte, F., McCarthy, J. R., Pitteri, S., Massoud, T. F., Paulmurugan, R. 2021: e2101387


    Polymeric nanocarriers (PNCs) can be used to deliver therapeutic microRNAs (miRNAs) to solid cancers. However, the ability of these nanocarriers to specifically target tumors remains a challenge. Alternatively, extracellular vesicles (EVs) derived from tumor cells show homotypic affinity to parent cells, but loading sufficient amounts of miRNAs into EVs is difficult. Here, we investigate whether uPAR-targeted delivery of nanococktails containing PNCs loaded with therapeutic antimiRNAs, and coated with uPA engineered extracellular vesicles (uPA-eEVs) can elicit synergistic antitumor responses. The uPA-eEVs coating on PNCs increases natural tumor targeting affinities, thereby enhancing the antitumor activity of antimiRNA nanococktails. The systemic administration of uPA-eEV-PNCs nanococktail showed a robust tumor tropism, which significantly enhanced the combinational antitumor effects of antimiRNA-21 and antimiRNA-10b, and led to significant tumor regression and extension of progression free survival for syngeneic 4T1 tumor-bearing mice. In addition, the uPA-eEV-PNCs-antimiRNAs nanococktail plus low dose doxorubicin resulted in a synergistic antitumor effect as evidenced by inhibition of tumor growth, reduction of lung metastases, and extension of survival of 4T1 tumor-bearing mice. Our targeted combinational nanococktail strategy could be readily translated to the clinical setting by using autologous cancer cells that have flexibility for ex vivo expansion and genetic engineering. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/adhm.202101387

    View details for PubMedID 34879180

  • Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms. Nature communications Yoshikawa, A. M., Rangel, A., Feagin, T., Chun, E. M., Wan, L., Li, A., Moekl, L., Wu, D., Eisenstein, M., Pitteri, S., Soh, H. T. 2021; 12 (1): 7106


    Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize the biological function of such modifications have been greatly hampered by the lack of affinity reagents that can differentiate protein glycoforms with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases, which are capable of recognizing and differentiating between specific protein glycoforms. Using this approach, we were able to select base-modified aptamers that exhibit strong selectivity for specific glycoforms of two different proteins. These aptamers can discriminate between molecules that differ only in their glycan modifications, and can also be used to label glycoproteins on the surface of cultured cells. We believe our strategy should offer a generally-applicable approach for developing useful reagents for glycobiology research.

    View details for DOI 10.1038/s41467-021-26933-1

    View details for PubMedID 34876561

  • Multi-omics analysis of spatially distinct stromal cells reveals tumor-induced O-glycosylation of the CDK4-pRB axis in fibroblasts at the invasive tumor edge. Cancer research Bouchard, G., Garcia-Marques, F. J., Karacosta, L. G., Zhang, W., Bermudez, A., Riley, N. M., Varma, S., Mehl, L. C., Benson, J. A., Shrager, J. B., Bertozzi, C. R., Pitteri, S. J., Giaccia, A. J., Plevritis, S. K. 2021


    The invasive leading edge represents a potential gateway for tumor metastasis. The role of fibroblasts from the tumor edge in promoting cancer invasion and metastasis has not been comprehensively elucidated. We hypothesize that crosstalk between tumor and stromal cells within the tumor microenvironment (TME) results in activation of key biological pathways depending on their position in the tumor (edge vs core). Here we highlight phenotypic differences between tumor-adjacent-fibroblasts (TAF) from the invasive edge and tumor core fibroblasts (TCF) from the tumor core, established from human lung adenocarcinomas. A multi-omics approach that includes genomics, proteomics, and O-glycoproteomics was used to characterize crosstalk between TAFs and cancer cells. These analyses showed that O-glycosylation, an essential post-translational modification resulting from sugar metabolism, alters key biological pathways including the cyclin-dependent kinase 4 and phosphorylated retinoblastoma protein (CDK4-pRB) axis in the stroma and indirectly modulates pro-invasive features of cancer cells. In summary, the O-glycoproteome represents a new consideration for important biological processes involved in tumor-stroma crosstalk and a potential avenue to improve the anti-cancer efficacy of CDK4 inhibitors.

    View details for DOI 10.1158/0008-5472.CAN-21-1705

    View details for PubMedID 34853070

  • Identifying a novel glycolytic inhibitor for treatment of aggressive prostate cancer. Stoyanova, T., Rice, M. A., Kumar, V., Tailor, D., Garcia-Marques, F., Bermudez, A., Kanchustambham, V., Shankar, V., Inde, Z., Pandrala, M., Nolley, R., Ghoochani, A., Liu, S., Aslan, M., Agarwal, A., Buckup, M., Hsu, E., Going, C. C., Peehl, D. M., Dixon, S. J., Zare, R. N., Brooks, J. D., Pitteri, S. J., Malhotra, S. V., Stoyanova, T. AMER ASSOC CANCER RESEARCH. 2021
  • Trop2 regulates prostate cancer growth and metastasis through distinct molecular mechanisms. Stoyanova, T., Hsu, E., Liu, S., Marques, F., Bermudez, A., Aslan, M., Shen, M., Pitteri, S., Brooks, J. D. AMER ASSOC CANCER RESEARCH. 2021
  • Lineage plasticity in small cell lung cancer generates non- neuroendocrine cells primed for vascular mimicry. Pearsall, S. M., Williamson, S. C., Marques, F., Humphrey, S., Hughes, E., Cannell, I., Frese, K. K., Galvin, M., Carter, M., Priest, L., Kerr, A., Humphries, M. J., Humphries, J. D., Blackhall, F., Hannon, G., Sage, J., Pitteri, S. J., Simpson, K. L., Dive, C. AMER ASSOC CANCER RESEARCH. 2021
  • A novel oncogene mediated metabolic gene signature predicts breast cancer outcome. Aslan, M., Hsu, E., Marques, F., Bermudez, A., Shen, M., Rice, M. A., Liu, S., West, R., Pitteri, S. J., Gyorffy, B., Stoyanova, T. AMER ASSOC CANCER RESEARCH. 2021
  • MAPK PATHWAY INHIBITION SENSITIZES TO IMMUNOTHERAPY IN BRAF-MUTANT GLIOMAS Park, J., Barrette, A., Wang, W., Grossauer, S., Grant, G., Lau, K., Pitteri, S., Monje, M., Petritsch, C. OXFORD UNIV PRESS INC. 2021: 3-4
  • Y box binding protein 1 inhibition as a targeted therapy for ovarian cancer. Cell chemical biology Tailor, D., Resendez, A., Garcia-Marques, F. J., Pandrala, M., Going, C. C., Bermudez, A., Kumar, V., Rafat, M., Nambiar, D. K., Honkala, A., Le, Q., Sledge, G. W., Graves, E., Pitteri, S. J., Malhotra, S. V. 2021


    Y box binding protein 1 (YB-1) is a multifunctional protein associated with tumor progression and the emergence of treatment resistance (TR). Here, we report an azopodophyllotoxin small molecule, SU056, that potently inhibits tumor growth and progression via YB-1 inhibition. This YB-1 inhibitor inhibits cell proliferation, resistance to apoptosis in ovarian cancer (OC) cells, and arrests in the G1 phase. Inhibitor treatment leads to enrichment of proteins associated with apoptosis and RNA degradation pathways while downregulating spliceosome pathway. Invivo, SU056 independently restrains OC progression and exerts a synergistic effect with paclitaxel to further reduce disease progression with no observable liver toxicity. Moreover, invitro mechanistic studies showed delayed disease progression via inhibition of drug efflux and multidrug resistance 1, and significantly lower neurotoxicity as compared with etoposide. These data suggest that YB-1 inhibition may be an effective strategy to reduce OC progression, antagonize TR, and decrease patient mortality.

    View details for DOI 10.1016/j.chembiol.2021.02.014

    View details for PubMedID 33713600

  • Discovery of CASP8 as a potential biomarker for high-risk prostate cancer through a high-multiplex immunoassay. Scientific reports Liu, S. n., Garcia-Marques, F. n., Zhang, C. A., Lee, J. J., Nolley, R. n., Shen, M. n., Hsu, E. C., Aslan, M. n., Koul, K. n., Pitteri, S. J., Brooks, J. D., Stoyanova, T. n. 2021; 11 (1): 7612


    Prostate cancer remains the most common non-cutaneous malignancy among men in the United States. To discover potential serum-based biomarkers for high-risk prostate cancer, we performed a high-multiplex immunoassay utilizing patient-matched pre-operative and post-operative serum samples from ten men with high-grade and high-volume prostate cancer. Our study identified six (CASP8, MSLN, FGFBP1, ICOSLG, TIE2 and S100A4) out of 174 proteins that were significantly decreased after radical prostatectomy. High levels of CASP8 were detected in pre-operative serum samples when compared to post-operative serum samples and serum samples from patients with benign prostate hyperplasia (BPH). By immunohistochemistry, CASP8 protein was expressed at higher levels in prostate cancer tissues compared to non-cancerous and BPH tissues. Likewise, CASP8 mRNA expression was significantly upregulated in prostate cancer when compared to benign prostate tissues in four independent clinical datasets. In addition, mRNA levels of CASP8 were higher in patients with recurrent prostate cancer when compared to patients with non-recurrent prostate cancer and high expression of CASP8 was associated with worse disease-free survival and overall survival in renal cancer. Together, our results suggest that CASP8 may potentially serve as a biomarker for high-risk prostate cancer and possibly renal cancer.

    View details for DOI 10.1038/s41598-021-87155-5

    View details for PubMedID 33828176

  • MCM2-7 complex is a novel druggable target for neuroendocrine prostate cancer. Scientific reports Hsu, E. C., Shen, M., Aslan, M., Liu, S., Kumar, M., Garcia-Marques, F., Nguyen, H. M., Nolley, R., Pitteri, S. J., Corey, E., Brooks, J. D., Stoyanova, T. 2021; 11 (1): 13305


    Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer that rarely develops de novo in primary tumors and is commonly acquired during the development of treatment resistance. NEPC is characterized by gain of neuroendocrine markers and loss of androgen receptor (AR), making it resistant to current therapeutic strategies targeting the AR signaling axis. Here, we report that MCM2, MCM3, MCM4, and MCM6 (MCM2/3/4/6) are elevated in human NEPC and high levels of MCM2/3/4/6 are associated with liver metastasis and poor survival in prostate cancer patients. MCM2/3/4/6 are four out of six proteins that form a core DNA helicase (MCM2-7) responsible for unwinding DNA forks during DNA replication. Inhibition of MCM2-7 by treatment with ciprofloxacin inhibits NEPC cell proliferation and migration in vitro, significantly delays NEPC tumor xenograft growth, and partially reverses the neuroendocrine phenotype in vivo. Our study reveals the clinical relevance of MCM2/3/4/6 proteins in NEPC and suggests that inhibition of MCM2-7 may represent a new therapeutic strategy for NEPC.

    View details for DOI 10.1038/s41598-021-92552-x

    View details for PubMedID 34172788

  • Oncogene-mediated metabolic gene signature predicts breast cancer outcome. NPJ breast cancer Aslan, M., Hsu, E. C., Garcia-Marques, F. J., Bermudez, A., Liu, S., Shen, M., West, M., Zhang, C. A., Rice, M. A., Brooks, J. D., West, R., Pitteri, S. J., Győrffy, B., Stoyanova, T. 2021; 7 (1): 141


    Breast cancer remains the second most lethal cancer among women in the United States and triple-negative breast cancer is the most aggressive subtype with limited treatment options. Trop2, a cell membrane glycoprotein, is overexpressed in almost all epithelial cancers. In this study, we demonstrate that Trop2 is overexpressed in triple-negative breast cancer (TNBC), and downregulation of Trop2 delays TNBC cell and tumor growth supporting the oncogenic role of Trop2 in breast cancer. Through proteomic profiling, we discovered a metabolic signature comprised of TALDO1, GPI, LDHA, SHMT2, and ADK proteins that were downregulated in Trop2-depleted breast cancer tumors. The identified oncogene-mediated metabolic gene signature is significantly upregulated in TNBC patients across multiple RNA-expression clinical datasets. Our study further reveals that the metabolic gene signature reliably predicts poor survival of breast cancer patients with early stages of the disease. Taken together, our study identified a new five-gene metabolic signature as an accurate predictor of breast cancer outcome.

    View details for DOI 10.1038/s41523-021-00341-6

    View details for PubMedID 34711841

  • Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography. Methods in molecular biology (Clifton, N.J.) Bermudez, A., Pitteri, S. J. 2021; 2271: 107–20


    Glycosylation is a biologically important and complex protein posttranslational modification. The emergence of glycoproteomic technologies to identify and characterize glycans on proteins has the potential to enable a better understanding the role of glycosylation in biology, disease states, and other areas of interest. In particular, the analysis of intact glycopeptides by mass spectrometry allows information about glycan location and composition to be ascertained. However, such analysis is often complicated by extensive glycan diversity and the low abundance of glycopeptides in a complex mixture relative to nonglycosylated peptides. Enrichment of glycopeptides from a protein enzymatic digest is an effective approach to overcome such challenges. In this chapter, we described a glycopeptide enrichment method combining strong anion exchange, electrostatic repulsion, and hydrophilic interaction chromatography (SAX-ERLIC). Following enzymatic digestion of proteins into peptides, SAX-ERLIC is performed by solid phase extraction to enrich glycopeptides from biological samples with subsequent LC-MS/MS analysis. Glycopeptide data generated using the SAX-ERLIC enrichment yields a high number of total and unique glycopeptide identifications which can be mapped back to proteins. The enrichment strategy is robust, easy to perform, and does not require cleavage of glycans prior to LC-MS/MS analysis.

    View details for DOI 10.1007/978-1-0716-1241-5_8

    View details for PubMedID 33908003

  • Plectin is a regulator of prostate cancer growth and metastasis. Oncogene Buckup, M., Rice, M. A., Hsu, E., Garcia-Marques, F., Liu, S., Aslan, M., Bermudez, A., Huang, J., Pitteri, S. J., Stoyanova, T. 2020


    Prostate cancer is responsible for over 30,000 US deaths annually, attributed largely to incurable metastatic disease. Here, we demonstrate that high levels of plectin are associated with localized and metastatic human prostate cancer when compared to benign prostate tissues. Knock-down of plectin inhibits prostate cancer cell growth and colony formation in vitro, and growth of prostate cancer xenografts in vivo. Plectin knock-down further impairs aggressive and invasive cellular behavior assessed by migration, invasion, and wound healing in vitro. Consistently, plectin knock-down cells have impaired metastatic colonization to distant sites including liver, lung, kidney, bone, and genitourinary system. Plectin knock-down inhibited number of metastases per organ, as well as decreased overall metastatic burden. To gain insights into the role of plectin in prostate cancer growth and metastasis, we performed proteomic analysis of prostate cancer plectin knock-down xenograft tissues. Gene set enrichment analysis shows an increase in levels of proteins involved with extracellular matrix and laminin interactions, and a decrease in levels of proteins regulating amino acid metabolism, cytoskeletal proteins, and cellular response to stress. Collectively these findings demonstrate that plectin is an important regulator of prostate cancer cell growth and metastasis.

    View details for DOI 10.1038/s41388-020-01557-9

    View details for PubMedID 33219316

  • Genomic analysis of Vascular Invasion in Hepatocellular Carcinoma (HCC) Reveals Molecular Drivers and Predictive Biomarkers. Hepatology (Baltimore, Md.) Krishnan, M. S., Rajan Kd, A. n., Park, J. n., Arjunan, V. n., Garcia Marques, F. J., Bermudez, A. n., Girvan, O. A., Hoang, N. S., Yin, J. n., Nguyen, M. H., Kothary, N. n., Pitteri, S. n., Felsher, D. W., Dhanasekaran, R. n. 2020


    Vascular invasion is a critical risk factor for hepatocellular carcinoma (HCC) recurrence and poor survival. The molecular drivers of vascular invasion in HCC are largely unknown. Deciphering the molecular landscape of invasive HCC will help identify novel therapeutic targets and noninvasive biomarkers. To this end, we undertook this study to evaluate the genomic, transcriptomic, and proteomic profile of tumors with vascular invasion using the multi-platform cancer genome atlas (TCGA) data (n=373). In the TCGA liver hepatocellular carcinoma (LIHC) cohort, macrovascular invasion was present in 5% (n=17) of tumors and microvascular invasion in 25% (n=94) of tumors. Functional pathway analysis revealed that the MYC oncogene was a common upstream regulator of the mRNA, miRNA and proteomic changes in vascular invasion. We performed comparative proteomic analyses of invasive human HCC and MYC driven murine HCC and identified fibronectin to be proteomic biomarker of invasive HCC (mouse Fn1 p= 1.7 X 10-11 ; human FN1 p=1.5 X 10-4 ) conserved across the two species. Mechanistically, we show that FN1 promotes the migratory and invasive phenotype of HCC cancer cells. We demonstrate tissue overexpression of fibronectin in human HCC using a large independent cohort of human HCC tissue microarray (n=153; p<0.001). Lastly, we showed that plasma fibronectin levels were significantly elevated in patients with HCC (n=35, mean=307.7 μg/ml, SEM=35.9) when compared to cirrhosis (n=10, mean=41.8 μg/ml, SEM=13.3; p<0.0001). CONCLUSION: Our study evaluates the molecular landscape of tumors with vascular invasion, identifying distinct transcriptional, epigenetic and proteomic changes driven by the MYC oncogene. We show that MYC upregulates fibronectin expression which promotes HCC invasiveness. In addition, we identify fibronectin to be a promising non-invasive proteomic biomarker of vascular invasion in HCC.

    View details for DOI 10.1002/hep.31614

    View details for PubMedID 33140851

  • Trop2 is a driver of metastatic prostate cancer with neuroendocrine phenotype via PARP1. Proceedings of the National Academy of Sciences of the United States of America Hsu, E. C., Rice, M. A., Bermudez, A. n., Marques, F. J., Aslan, M. n., Liu, S. n., Ghoochani, A. n., Zhang, C. A., Chen, Y. S., Zlitni, A. n., Kumar, S. n., Nolley, R. n., Habte, F. n., Shen, M. n., Koul, K. n., Peehl, D. M., Zoubeidi, A. n., Gambhir, S. S., Kunder, C. A., Pitteri, S. J., Brooks, J. D., Stoyanova, T. n. 2020


    Resistance to androgen deprivation therapy, or castration-resistant prostate cancer (CRPC), is often accompanied by metastasis and is currently the ultimate cause of prostate cancer-associated deaths in men. Recently, secondary hormonal therapies have led to an increase of neuroendocrine prostate cancer (NEPC), a highly aggressive variant of CRPC. Here, we identify that high levels of cell surface receptor Trop2 are predictive of recurrence of localized prostate cancer. Moreover, Trop2 is significantly elevated in CRPC and NEPC, drives prostate cancer growth, and induces neuroendocrine phenotype. Overexpression of Trop2 induces tumor growth and metastasis while loss of Trop2 suppresses these abilities in vivo. Trop2-driven NEPC displays a significant up-regulation of PARP1, and PARP inhibitors significantly delay tumor growth and metastatic colonization and reverse neuroendocrine features in Trop2-driven NEPC. Our findings establish Trop2 as a driver and therapeutic target for metastatic prostate cancer with neuroendocrine phenotype and suggest that high Trop2 levels could identify cancers that are sensitive to Trop2-targeting therapies and PARP1 inhibition.

    View details for DOI 10.1073/pnas.1905384117

    View details for PubMedID 31932422

  • A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry-Based Glycoproteomics. Molecular & cellular proteomics : MCP Riley, N. M., Bertozzi, C. R., Pitteri, S. J. 2020; 20: 100029


    Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography and its derivatives, porous graphitic carbon, reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as mass spectrometry instrumentation and software improve, so this review aims to help equip researchers with the necessary information to choose appropriate enrichment strategies that best complement these efforts.

    View details for DOI 10.1074/mcp.R120.002277

    View details for PubMedID 33583771

  • Discovery of PTN as a serum-based biomarker of pro-metastatic prostate cancer. British journal of cancer Liu, S. n., Shen, M. n., Hsu, E. C., Zhang, C. A., Garcia-Marques, F. n., Nolley, R. n., Koul, K. n., Rice, M. A., Aslan, M. n., Pitteri, S. J., Massie, C. n., George, A. n., Brooks, J. D., Gnanapragasam, V. J., Stoyanova, T. n. 2020


    Distinguishing clinically significant from indolent prostate cancer (PC) is a major clinical challenge. We utilised targeted protein biomarker discovery approach to identify biomarkers specific for pro-metastatic PC. Serum samples from the cancer-free group; Cambridge Prognostic Group 1 (CPG1, low risk); CPG5 (high risk) and metastatic disease were analysed using Olink Proteomics panels. Tissue validation was performed by immunohistochemistry in a radical prostatectomy cohort (n = 234). We discovered that nine proteins (pleiotrophin (PTN), MK, PVRL4, EPHA2, TFPI-2, hK11, SYND1, ANGPT2, and hK14) were elevated in metastatic PC patients when compared to other groups. PTN levels were increased in serum from men with CPG5 compared to benign and CPG1. High tissue PTN level was an independent predictor of biochemical recurrence and metastatic progression in low- and intermediate-grade disease. These findings suggest that PTN may represent a novel biomarker for the presence of poor prognosis local disease with the potential to metastasise warranting further investigation.

    View details for DOI 10.1038/s41416-020-01200-0

    View details for PubMedID 33288843

  • Reconstructed Apoptotic Bodies as Targeted "Nano Decoys" to Treat Intracellular Bacterial Infections within Macrophages and Cancer Cells. ACS nano Bose, R. J., Tharmalingam, N. n., Garcia Marques, F. J., Sukumar, U. K., Natarajan, A. n., Zeng, Y. n., Robinson, E. n., Bermudez, A. n., Chang, E. n., Habte, F. n., Pitteri, S. J., McCarthy, J. R., Gambhir, S. S., Massoud, T. F., Mylonakis, E. n., Paulmurugan, R. n. 2020


    Staphylococcus aureus (S. aureus) is a highly pathogenic facultative anaerobe that in some instances resides as an intracellular bacterium within macrophages and cancer cells. This pathogen can establish secondary infection foci, resulting in recurrent systemic infections that are difficult to treat using systemic antibiotics. Here, we use reconstructed apoptotic bodies (ReApoBds) derived from cancer cells as "nano decoys" to deliver vancomycin intracellularly to kill S. aureus by targeting inherent "eat me" signaling of ApoBds. We prepared ReApoBds from different cancer cells (SKBR3, MDA-MB-231, HepG2, U87-MG, and LN229) and used them for vancomycin delivery. Physicochemical characterization showed ReApoBds size ranges from 80 to 150 nm and vancomycin encapsulation efficiency of 60 ± 2.56%. We demonstrate that the loaded vancomycin was able to kill intracellular S. aureus efficiently in an in vitro model of S. aureus infected RAW-264.7 macrophage cells, and U87-MG (p53-wt) and LN229 (p53-mt) cancer cells, compared to free-vancomycin treatment (P < 0.001). The vancomycin loaded ReApoBds treatment in S. aureus infected macrophages showed a two-log-order higher CFU reduction than the free-vancomycin treatment group. In vivo studies revealed that ReApoBds can specifically target macrophages and cancer cells. Vancomycin loaded ReApoBds have the potential to kill intracellular S. aureus infection in vivo in macrophages and cancer cells.

    View details for DOI 10.1021/acsnano.0c00921

    View details for PubMedID 32347709

  • Novel Aza-podophyllotoxin derivative induces oxidative phosphorylation and cell death via AMPK activation in triple-negative breast cancer. British journal of cancer Tailor, D. n., Going, C. C., Resendez, A. n., Kumar, V. n., Nambiar, D. K., Li, Y. n., Dheeraj, A. n., LaGory, E. L., Ghoochani, A. n., Birk, A. M., Stoyanova, T. n., Ye, J. n., Giaccia, A. J., Le, Q. T., Singh, R. P., Sledge, G. W., Pitteri, S. J., Malhotra, S. V. 2020


    To circumvent Warburg effect, several clinical trials for different cancers are utilising a combinatorial approach using metabolic reprogramming and chemotherapeutic agents including metformin. The majority of these metabolic interventions work via indirectly activating AMP-activated protein kinase (AMPK) to alter cellular metabolism in favour of oxidative phosphorylation over aerobic glycolysis. The effect of these drugs is dependent on glycaemic and insulin conditions.  Therefore, development of small molecules, which can activate AMPK, irrespective of the energy state, may be a better approach for triple-negative breast cancer (TNBC) treatment.Therapeutic effect of SU212 on TNBC cells was examined using in vitro and in vivo models.We developed and characterised the efficacy of novel AMPK activator (SU212) that selectively induces oxidative phosphorylation and decreases glycolysis in TNBC cells, while not affecting these pathways in normal cells.   SU212 accomplished this metabolic reprogramming by activating AMPK independent of energy stress and irrespective of the glycaemic/insulin state. This leads to mitotic phase arrest and apoptosis in TNBC cells. In vivo, SU212 inhibits tumour growth, cancer progression and metastasis.SU212 directly activates AMPK in TNBC cells, but does not hamper glucose metabolism in normal cells. Our study provides compelling preclinical data for further development of SU212 for the treatment of TNBC.

    View details for DOI 10.1038/s41416-020-01137-4

    View details for PubMedID 33139797

  • Novel glycolysis inhibitor improves the therapeutic regimen for triple negative breast cancer under hyperglycemic condition Tailor, D., Kumar, V., Resendez, A., Going, C., Pitteri, S., Malhotra, S. AMER CHEMICAL SOC. 2019
  • LARP1 binding to hepatitis C virus particles is correlated with intracellular retention of viral infectivity. Virus research Plissonnier, M., Cottarel, J., Piver, E., Kullolli, M., Centonze, F. G., Pitteri, S., Farhan, H., Meunier, J., Zoulim, F., Parent, R. 2019: 197679


    Hepatitis C virus (HCV) virions contain a subset of host liver cells proteome often composed of interesting virus-interacting factors. A proteomic analysis performed on double gradient-purified clinical HCV highlighted the translation regulator LARP1 on these virions. This finding was validated using post-virion capture and immunoelectron microscopy, immunoprecipitation applied to in vitro (Huh7.5 liver cells) grown (Gt2a, JFH1 strain) and patient-derived (Gt1a) HCV particles. Upon HCV infection of Huh7.5 cells, we observed a drastic transfer of LARP1 to lipid droplets, inducing colocalization with core proteins. RNAi-mediated depletion of LARP1 using the C911 control approach decreased extracellular infectivity of HCV Gt1a (H77), Gt2a (JFH1), and Gt3a (S52 chimeric strain), yet increased their intracellular infectivity. This latter effect was unrelated to changes in the hepatocyte secretory pathway, as evidenced using a functional RUSH assay. These results indicate that LARP1 binds to HCV, an event associated with retention of intracellular infectivity.

    View details for DOI 10.1016/j.virusres.2019.197679

    View details for PubMedID 31398365

  • Cancer specific caloric restriction using novel small molecule improves the therapeutic regime for triple negative breast cancer Tailor, D., Resendez, A., Kumar, V., Going, C., Pitteri, S., Malhotra, S. AMER ASSOC CANCER RESEARCH. 2019
  • Proteomic Identification and Time-Course Monitoring of Secreted Proteins During Expansion of Human Mesenchymal Stem/Stromal in Stirred-Tank Bioreactor. Frontiers in bioengineering and biotechnology Mizukami, A. n., Thomé, C. H., Ferreira, G. A., Lanfredi, G. P., Covas, D. T., Pitteri, S. J., Swiech, K. n., Faça, V. M. 2019; 7: 154


    The therapeutic potential of mesenchymal stem/stromal cells (MSC) is widely recognized for the treatment of several diseases, including acute graft-vs.-host disease (GVHD), hematological malignancies, cardiovascular, bone, and cartilage diseases. More recently, this therapeutic efficacy has been attributed to the bioactive molecules that these cells secrete (secretome), now being referred as medicinal signaling cells. This fact raises the opportunity of therapeutically using MSC-derived soluble factors rather than cells themselves, enabling their translation into the clinic. Indeed, many clinical trials are now studying the effects of MSC-secretome in the context of cell-free therapy. MSC secretome profile varies between donors, source, and culture conditions, making their therapeutic use very challenging. Therefore, identifying these soluble proteins and evaluating their production in a reproducible and scalable manner is even more relevant. In this work, we analyzed the global profile of proteins secreted by umbilical cord matrix (UCM) derived-MSC in static conditions by using mass spectrometry, enabling the identification of thousands of proteins. Afterwards, relevant proteins were chosen and monitored in the supernatant of a fully-controllable, closed and scalable system (bioreactor) by using multiple reaction monitoring (MRM) mass spectrometric technique in a time-dependent manner. The results showed that the majority of interesting proteins were enriched through time in culture, with the last day of culture being the ideal time for supernatant collection. The use of this regenerative "soup," which is frequently discarded, could represent a step toward a safe, robust and reproducible cell-free product to be used in the medical therapeutic field. The future use of chemically defined culture-media will certainly facilitate secretome production according to Good Manufacturing Practice (GMP) standards.

    View details for DOI 10.3389/fbioe.2019.00154

    View details for PubMedID 31297369

    View details for PubMedCentralID PMC6607109

  • Honey bee Royalactin unlocks conserved pluripotency pathway in mammals. Nature communications Wan, D. C., Morgan, S. L., Spencley, A. L., Mariano, N., Chang, E. Y., Shankar, G., Luo, Y., Li, T. H., Huh, D., Huynh, S. K., Garcia, J. M., Dovey, C. M., Lumb, J., Liu, L., Brown, K. V., Bermudez, A., Luong, R., Zeng, H., Mascetti, V. L., Pitteri, S. J., Wang, J., Tu, H., Quarta, M., Sebastiano, V., Nusse, R., Rando, T. A., Carette, J. E., Bazan, J. F., Wang, K. C. 2018; 9 (1): 5078


    Royal jelly is the queen-maker for the honey bee Apis mellifera, and has cross-species effects on longevity, fertility, and regeneration in mammals. Despite this knowledge, how royal jelly or its components exert their myriad effects has remained poorly understood. Using mouse embryonic stem cells as a platform, here we report that through its major protein component Royalactin, royal jelly can maintain pluripotency by activating a ground-state pluripotency-like gene network. We further identify Regina, a mammalian structural analog of Royalactin that also induces a naive-like state in mouse embryonic stem cells. This reveals an important innate program for stem cell self-renewal with broad implications in understanding the molecular regulation of stem cell fate across species.

    View details for PubMedID 30510260

  • Analysis of Released N-Glycans and Glycopeptide Profiling of Prostate Cancer Tissue Totten, S. M., Bermudez, A., Guerrero, A., Yan, J., Jones, A., Brooks, J. D., Pitteri, S. J. OXFORD UNIV PRESS INC. 2018: 1056
  • Making Glycoproteomics via Mass Spectrometry More Accessible to the greater Scientific Community Driessen, M. D., Going, C. C., Woo, C. M., Pitteri, S. J., Bertozzi, C. R. OXFORD UNIV PRESS INC. 2018: 1013
  • Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents ACS NANO Bose, R. C., Kumar, S., Zeng, Y., Afjei, R., Robinson, E., Lau, K., Bermudez, A., Habte, F., Pitteri, S. J., Sinclair, R., Willmann, J. K., Massoud, T. F., Gambhir, S. S., Paulmurugan, R. 2018; 12 (11): 10817–32
  • Quantitative Proteomic Profiling Reveals Key Pathways in the Anticancer Action of Methoxychalcone Derivatives in Triple Negative Breast Cancer JOURNAL OF PROTEOME RESEARCH Going, C. C., Tailor, D., Kumar, V., Birk, A. M., Pandrala, M., Rice, M. A., Stoyanova, T., Malhotra, S., Pitteri, S. J. 2018; 17 (10): 3574–85
  • CRISPR-Mediated Reorganization of Chromatin Loop Structure. Journal of visualized experiments : JoVE Morgan, S. L., Chang, E. Y., Mariano, N. C., Bermudez, A., Arruda, N. L., Wu, F., Luo, Y., Shankar, G., Huynh, S. K., Huang, C., Pitteri, S. J., Wang, K. C. 2018


    Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

    View details for PubMedID 30272647

  • Integrative Personal Omics Profiles during Periods of Weight Gain and Loss. Cell systems Piening, B. D., Zhou, W. n., Contrepois, K. n., Röst, H. n., Gu Urban, G. J., Mishra, T. n., Hanson, B. M., Bautista, E. J., Leopold, S. n., Yeh, C. Y., Spakowicz, D. n., Banerjee, I. n., Chen, C. n., Kukurba, K. n., Perelman, D. n., Craig, C. n., Colbert, E. n., Salins, D. n., Rego, S. n., Lee, S. n., Zhang, C. n., Wheeler, J. n., Sailani, M. R., Liang, L. n., Abbott, C. n., Gerstein, M. n., Mardinoglu, A. n., Smith, U. n., Rubin, D. L., Pitteri, S. n., Sodergren, E. n., McLaughlin, T. L., Weinstock, G. M., Snyder, M. P. 2018


    Advances in omics technologies now allow an unprecedented level of phenotyping for human diseases, including obesity, in which individual responses to excess weight are heterogeneous and unpredictable. To aid the development of better understanding of these phenotypes, we performed a controlled longitudinal weight perturbation study combining multiple omics strategies (genomics, transcriptomics, multiple proteomics assays, metabolomics, and microbiomics) during periods of weight gain and loss in humans. Results demonstrated that: (1) weight gain is associated with the activation of strong inflammatory and hypertrophic cardiomyopathy signatures in blood; (2) although weight loss reverses some changes, a number of signatures persist, indicative of long-term physiologic changes; (3) we observed omics signatures associated with insulin resistance that may serve as novel diagnostics; (4) specific biomolecules were highly individualized and stable in response to perturbations, potentially representing stable personalized markers. Most data are available open access and serve as a valuable resource for the community.

    View details for PubMedID 29361466

  • Multi-lectin Affinity Chromatography and Quantitative Proteomic Analysis Reveal Differential Glycoform Levels between Prostate Cancer and Benign Prostatic Hyperplasia Sera. Scientific reports Totten, S. M., Adusumilli, R. n., Kullolli, M. n., Tanimoto, C. n., Brooks, J. D., Mallick, P. n., Pitteri, S. J. 2018; 8 (1): 6509


    Currently prostate-specific antigen is used for prostate cancer (PCa) screening, however it lacks the necessary specificity for differentiating PCa from other diseases of the prostate such as benign prostatic hyperplasia (BPH), presenting a clinical need to distinguish these cases at the molecular level. Protein glycosylation plays an important role in a number of cellular processes involved in neoplastic progression and is aberrant in PCa. In this study, we systematically interrogate the alterations in the circulating levels of hundreds of serum proteins and their glycoforms in PCa and BPH samples using multi-lectin affinity chromatography and quantitative mass spectrometry-based proteomics. Specific lectins (AAL, PHA-L and PHA-E) were used to target and chromatographically separate core-fucosylated and highly-branched protein glycoforms for analysis, as differential expression of these glycan types have been previously associated with PCa. Global levels of CD5L, CFP, C8A, BST1, and C7 were significantly increased in the PCa samples. Notable glycoform-specific alterations between BPH and PCa were identified among proteins CD163, C4A, and ATRN in the PHA-L/E fraction and among C4BPB and AZGP1 glycoforms in the AAL fraction. Despite these modest differences, substantial similarities in glycoproteomic profiles were observed between PCa and BPH sera.

    View details for PubMedID 29695737

  • How many human proteoforms are there? Nature chemical biology Aebersold, R. n., Agar, J. N., Amster, I. J., Baker, M. S., Bertozzi, C. R., Boja, E. S., Costello, C. E., Cravatt, B. F., Fenselau, C. n., Garcia, B. A., Ge, Y. n., Gunawardena, J. n., Hendrickson, R. C., Hergenrother, P. J., Huber, C. G., Ivanov, A. R., Jensen, O. N., Jewett, M. C., Kelleher, N. L., Kiessling, L. L., Krogan, N. J., Larsen, M. R., Loo, J. A., Ogorzalek Loo, R. R., Lundberg, E. n., MacCoss, M. J., Mallick, P. n., Mootha, V. K., Mrksich, M. n., Muir, T. W., Patrie, S. M., Pesavento, J. J., Pitteri, S. J., Rodriguez, H. n., Saghatelian, A. n., Sandoval, W. n., Schlüter, H. n., Sechi, S. n., Slavoff, S. A., Smith, L. M., Snyder, M. P., Thomas, P. M., Uhlén, M. n., Van Eyk, J. E., Vidal, M. n., Walt, D. R., White, F. M., Williams, E. R., Wohlschlager, T. n., Wysocki, V. H., Yates, N. A., Young, N. L., Zhang, B. n. 2018; 14 (3): 206–14


    Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

    View details for PubMedID 29443976

  • Mapping and quantification of over 2,000 O-linked glycopeptides in activated human T cells with isotope-targeted glycoproteomics (IsoTaG). Molecular & cellular proteomics : MCP Woo, C. M., Lund, P. J., Huang, A. C., Davis, M. M., Bertozzi, C. R., Pitteri, S. n. 2018


    Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example.  Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation.  Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites.  Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics.  This approach led to the identification of 2,219 intact O-linked glycopeptides across 1,045 glycoproteins.  A significant proportion (>45%) of the identified O-GlcNAc sites lie in close proximity to or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk.  Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence.  To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups.  Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells.  Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.

    View details for PubMedID 29351928

  • Making Glycoproteomics via Mass Spectrometry More Accessible to the greater Scientific Community Driessen, M. D., Going, C. C., Woo, C. M., Pitteri, S. J., Bertozzi, C. R. OXFORD UNIV PRESS INC. 2017: 1212
  • Assessing biological and technological variability in protein levels measured in pre-diagnostic plasma samples of women with breast cancer. Biomarker research Yeh, C. Y., Adusumilli, R., Kullolli, M., Mallick, P., John, E. M., Pitteri, S. J. 2017; 5: 30


    Quantitative proteomics allows for the discovery and functional investigation of blood-based pre-diagnostic biomarkers for early cancer detection. However, a major limitation of proteomic investigations in biomarker studies remains the biological and technical variability in the analysis of complex clinical samples. Moreover, unlike 'omics analogues such as genomics and transcriptomics, proteomics has yet to achieve reproducibility and long-term stability on a unified technological platform. Few studies have thoroughly investigated protein variability in pre-diagnostic samples of cancer patients across multiple platforms.We obtained ten blood plasma "case" samples collected up to 2 years prior to breast cancer diagnosis. Each case sample was paired with a matched control plasma from a full biological sister without breast cancer. We measured protein levels using both mass-spectrometry and antibody-based technologies to: (1) assess the technical considerations in different protein assays when analyzing limited clinical samples, and (2) evaluate the statistical power of potential diagnostic analytes.Although we found inherent technical variation in the three assays used, we detected protein dependent biological signal from the limited samples. The three assay types yielded 32 proteins with statistically significantly (p < 1E-01) altered expression levels between cases and controls, with no proteins retaining statistical significance after false discovery correction.Technical, practical, and study design considerations are essential to maximize information obtained in limited pre-diagnostic samples of cancer patients. This study provides a framework that estimates biological effect sizes critical for consideration in designing studies for pre-diagnostic blood-based biomarker detection.

    View details for DOI 10.1186/s40364-017-0110-y

    View details for PubMedID 29075496

    View details for PubMedCentralID PMC5645980

  • Characterization of Glycoproteins by Top Down UVPD Analysis Going, C., Huguet, R., Ferrer, D., Zabrouskov, V., Huhmer, A. R., Pitteri, S. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2017: S43
  • Parallel Comparison of N-Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC. Journal of proteome research Totten, S. M., Feasley, C. L., Bermudez, A., Pitteri, S. J. 2017; 16 (3): 1249-1260


    Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liquid chromatography using strong anion exchange solid-phase extraction (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was observed in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were additionally analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these experiments, 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation analysis in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.

    View details for DOI 10.1021/acs.jproteome.6b00849

    View details for PubMedID 28199111

  • Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes. Journal of proteome research Woo, C. M., Felix, A., Byrd, W. E., Zuegel, D. K., Ishihara, M., Azadi, P., Iavarone, A. T., Pitteri, S. J., Bertozzi, C. R. 2017


    Protein glycosylation can have an enormous variety of biological consequences, reflecting the molecular diversity encoded in glycan structures. This same structural diversity has imposed major challenges on the development of methods to study the intact glycoproteome. We recently introduced a method termed isotope-targeted glycoproteomics (IsoTaG), which utilizes isotope recoding to characterize azidosugar-labeled glycopeptides bearing fully intact glycans. Here, we describe the broad application of the method to analyze glycoproteomes from a collection of tissue-diverse cell lines. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. Application of the IsoTaG platform to 15 cell lines metabolically labeled with Ac4GalNAz or Ac4ManNAz revealed 1375 N- and 2159 O-glycopeptides, variously modified with 74 discrete glycan structures. Glycopeptide-bound glycans observed by IsoTaG were found to be comparable to released N-glycans identified by permethylation analysis. IsoTaG is therefore positioned to enhance structural understanding of the glycoproteome.

    View details for DOI 10.1021/acs.jproteome.6b01053

    View details for PubMedID 28244757

  • Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Nature communications Morgan, S. L., Mariano, N. C., Bermudez, A. n., Arruda, N. L., Wu, F. n., Luo, Y. n., Shankar, G. n., Jia, L. n., Chen, H. n., Hu, J. F., Hoffman, A. R., Huang, C. C., Pitteri, S. J., Wang, K. C. 2017; 8: 15993


    Chromatin looping is key to gene regulation, yet no broadly applicable methods to selectively modify chromatin loops have been described. We have engineered a method for chromatin loop reorganization using CRISPR-dCas9 (CLOuD9) to selectively and reversibly establish chromatin loops. We demonstrate the power of this technology to selectively modulate gene expression at targeted loci.

    View details for PubMedID 28703221

  • Vitamin D supplementation decreases serum 27-hydroxycholesterol in a pilot breast cancer trial. Breast cancer research and treatment Going, C. C., Alexandrova, L. n., Lau, K. n., Yeh, C. Y., Feldman, D. n., Pitteri, S. J. 2017


    27-hydroxycholesterol (27HC), an endogenous selective estrogen receptor modulator (SERM), drives the growth of estrogen receptor-positive (ER+) breast cancer. 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, is known to inhibit expression of CYP27B1, which is very similar in structure and function to CYP27A1, the synthesizing enzyme of 27HC. Therefore, we hypothesized that 1,25(OH)2D may also inhibit expression of CYP27A1, thereby reducing 27HC concentrations in the blood and tissues that express CYP27A1, including breast cancer tissue.27HC, 25-hydroxyvitamin D (25OHD), and 1,25(OH)2D were measured in sera from 29 breast cancer patients before and after supplementation with low-dose (400 IU/day) or high-dose (10,000 IU/day) vitamin D in the interval between biopsy and surgery.A significant increase (p = 4.3E-5) in 25OHD and a decrease (p = 1.7E-1) in 27HC was observed in high-dose versus low-dose vitamin D subjects. Excluding two statistical outliers, 25OHD and 27HC levels were inversely correlated (p = 7.0E-3).Vitamin D supplementation can decrease circulating 27HC of breast cancer patients, likely by CYP27A1 inhibition. This suggests a new and additional modality by which vitamin D can inhibit ER+ breast cancer growth, though a larger study is needed for verification.

    View details for PubMedID 29116467

  • Multi-Lectin Affinity Chromatography for Separation, Identification, and Quantitation of Intact Protein Glycoforms in Complex Biological Mixtures. Methods in molecular biology (Clifton, N.J.) Totten, S. M., Kullolli, M., Pitteri, S. J. 2017; 1550: 99-113


    Protein glycosylation is considered to be one of the most abundant post-translational modifications and is recognized for playing key roles in cellular functions. Aberrant N-linked glycosylation has been associated with several human diseases and has prompted the development and constant improvement of analytical tools to separate, characterize, and quantify glycoproteins in complex mixtures extracted from various biological samples (such as blood and tissue). Lectins, or carbohydrate-binding proteins, have been used as valuable tools for enriching for glycoproteins and selecting for specific types of glycosylation. Herein a method using multidimensional intact protein fractionation and LC-MS/MS analysis is described. Immunodepletion is used to remove highly abundant proteins from human plasma, followed by glycoform separation using multi-lectin affinity chromatography, in which specific lectins are chosen to capture and elute specific types of glycosylation. Reversed-phase chromatography prior to digestion is used for further fractionation, allowing for an increased number of protein identifications of moderate- to low-abundant proteins detectable in plasma. This method also incorporates isotopic labeling during alkylation for relative quantitation between two samples (such as a case and control). A bottom-up, tandem mass spectrometry-based proteomics approach is used for protein identification and quantitation, and allows for screening glycoform-specific changes across hundreds of plasma proteins.

    View details for DOI 10.1007/978-1-4939-6747-6_9

    View details for PubMedID 28188526

  • The Exosome Total Isolation Chip. ACS nano Liu, F. n., Vermesh, O. n., Mani, V. n., Ge, T. J., Madsen, S. J., Sabour, A. n., Hsu, E. C., Gowrishankar, G. n., Kanada, M. n., Jokerst, J. V., Sierra, R. G., Chang, E. n., Lau, K. n., Sridhar, K. n., Bermudez, A. n., Pitteri, S. J., Stoyanova, T. n., Sinclair, R. n., Nair, V. S., Gambhir, S. S., Demirci, U. n. 2017


    Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the device's broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 μL) of blood.

    View details for DOI 10.1021/acsnano.7b04878

    View details for PubMedID 29090896

  • Activation of Notch1 synergizes with multiple pathways in promoting castration-resistant prostate cancer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Stoyanova, T., Riedinger, M., Lin, S., Faltermeier, C. M., Smith, B. A., Zhang, K. X., Going, C. C., Goldstein, A. S., Lee, J. K., Drake, J. M., Rice, M. A., Hsu, E., Nowroozizadeh, B., Castor, B., Orellana, S. Y., Blum, S. M., Cheng, D., Pienta, K. J., Reiter, R. E., Pitteri, S. J., Huang, J., Witte, O. N. 2016; 113 (42): E6457-E6466


    Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.

    View details for DOI 10.1073/pnas.1614529113

    View details for PubMedID 27694579

  • Proteomic Analysis of Epithelial to Mesenchymal Transition (EMT) Reveals Cross-talk between SNAIL and HDAC1 Proteins in Breast Cancer Cells. Molecular & cellular proteomics Palma, C. d., Grassi, M. L., Thomé, C. H., Ferreira, G. A., Albuquerque, D., Pinto, M. T., Ferreira Melo, F. U., Kashima, S., Covas, D. T., Pitteri, S. J., Faça, V. M. 2016; 15 (3): 906-917


    Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-β, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.

    View details for DOI 10.1074/mcp.M115.052910

    View details for PubMedID 26764010

    View details for PubMedCentralID PMC4813709

  • FIG4 is a hepatitis C virus particle-bound protein implicated in virion morphogenesis and infectivity with cholesteryl ester modulation potential. journal of general virology Cottarel, J., Plissonnier, M., Kullolli, M., Pitteri, S., Clément, S., Millarte, V., Si-Ahmed, S., Farhan, H., Zoulim, F., Parent, R. 2016; 97 (1): 69-81


    There is growing evidence that virus particles also contain host cell proteins, which provide viruses with certain properties required for entry and release. A proteomic analysis performed on double gradient-purified hepatitis C virus from two highly viremic patients identified the Phosphatidylinositol 3,5-bisphosphate 5-phosphatase FIG4 (KIAA0274) as part of the viral particles. We validated the association using immunoelectron microscopy, immunoprecipitation and neutralization assays in vitro as well as patient-derived virus particles. RNAi-mediated reduction of FIG4 expression decreased cholesteryl ester (CE) levels along with intra- and extracellular viral infectivity without affecting HCV RNA levels. Likewise, overexpressing FIG4 increased intracellular CE levels as well as intra- and extracellular viral infectivity without affecting viral RNA levels. Triglyceride (TG) levels and lipid droplets (LD) parameters remained unaffected. The 3,5-bisphosphate 5-phosphatase active site of FIG4 was found to strongly condition these results. While FIG4 was found to localize to areas corresponding to viral assembly sites, at the immediate vicinity of LDs in calnexin+ and HCV core+ regions, no implication of FIG4 in the secretory pathway of the hepatocytes could be found using either FIG4 null mice, in vitro morphometry or functional assays of the ERGIC/Golgi compartments. This indicates that FIG4-dependent modulation of HCV infectivity is unrelated to alterations in the functionality of the secretory pathway. Because of the documented implication of CE in the composition and infectivity of HCV particles, these results suggest that FIG4 binds to HCV and modulates particle formation in a CE-related manner.

    View details for DOI 10.1099/jgv.0.000331

    View details for PubMedID 26519381

  • 3-D tumor models. Materials today Asghar, W., El Assal, R., Shafiee, H., Pitteri, S., Paulmurugan, R., Demirci, U. 2015; 18 (10): 539-553


    The natural microenvironment of tumors is composed of extracellular matrix (ECM), blood vasculature, and supporting stromal cells. The physical characteristics of ECM as well as the cellular components play a vital role in controlling cancer cell proliferation, apoptosis, metabolism, and differentiation. To mimic the tumor microenvironment outside the human body for drug testing, two-dimensional (2-D) and murine tumor models are routinely used. Although these conventional approaches are employed in preclinical studies, they still present challenges. For example, murine tumor models are expensive and difficult to adopt for routine drug screening. On the other hand, 2-D in vitro models are simple to perform, but they do not recapitulate natural tumor microenvironment, because they do not capture important three-dimensional (3-D) cell-cell, cell-matrix signaling pathways, and multi-cellular heterogeneous components of the tumor microenvironment such as stromal and immune cells. The three-dimensional (3-D) in vitro tumor models aim to closely mimic cancer microenvironments and have emerged as an alternative to routinely used methods for drug screening. Herein, we review recent advances in 3-D tumor model generation and highlight directions for future applications in drug testing.

    View details for DOI 10.1016/j.mattod.2015.05.002

    View details for PubMedID 28458612

  • In-depth quantitative analysis of protein glycoforms in human prostate cancer plasma Totten, S. M., Kulloli, M., Tanimoto, C., Brooks, J. D., Pitteri, S. J. AMER ASSOC CANCER RESEARCH. 2015
  • Glycoproteomic analysis of breast cancer cell lines for biomarker discovery Arampatzidou, M., Kullolli, M., Pitteri, S. J. AMER ASSOC CANCER RESEARCH. 2014
  • Intact MicroRNA Analysis Using High Resolution Mass Spectrometry JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Kullolli, M., Knouf, E., Arampatzidou, M., Tewari, M., Pitteri, S. J. 2014; 25 (1): 80-87


    MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that post-transcriptionally regulate gene expression, and play key roles in the regulation of a variety of cellular processes and in disease. New tools to analyze miRNAs will add understanding of the physiological origins and biological functions of this class of molecules. In this study, we investigate the utility of high resolution mass spectrometry for the analysis of miRNAs through proof-of-concept experiments. We demonstrate the ability of mass spectrometry to resolve and separate miRNAs and corresponding 3' variants in mixtures. The mass accuracy of the monoisotopic deprotonated peaks from various miRNAs is in the low ppm range. We compare fragmentation of miRNA by collision-induced dissociation (CID) and by higher-energy collisional dissociation (HCD) which yields similar sequence coverage from both methods but additional fragmentation by HCD versus CID. We measure the linear dynamic range, limit of detection, and limit of quantitation of miRNA loaded onto a C18 column. Lastly, we explore the use of data-dependent acquisition of MS/MS spectra of miRNA during online LC-MS and demonstrate that multiple charge states can be fragmented, yielding nearly full sequence coverage of miRNA on a chromatographic time scale. We conclude that high resolution mass spectrometry allows the separation and measurement of miRNAs in mixtures and a standard LC-MS setup can be adapted for online analysis of these molecules.

    View details for DOI 10.1007/s13361-013-0759-x

    View details for Web of Science ID 000329239600010

    View details for PubMedID 24174127

  • Performance evaluation of affinity ligands for depletion of abundant plasma proteins. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Kullolli, M., Warren, J., Arampatzidou, M., Pitteri, S. J. 2013; 939: 10-16


    Human plasma is a commonly used diagnostic fluid in clinical chemistry. In-depth plasma proteomic analysis is performed to search for disease biomarkers, however the large dynamic range of protein abundance in plasma presents a substantial analytical challenge. Removal of abundant plasma proteins using antibody capture approaches is a common and attractive means to reduce sample complexity and to aid the analysis of lower abundance proteins of interest. A novel class of heavy chain camelid-derived affinity ligands produced in Saccharomyces cerevisiae, has recently been developed as an alternative to antibody-based depletion methods. Here, we evaluate the performance characteristics of these ligands for removal of high abundance plasma proteins. Affinity ligands were tested for the removal of 14 abundant human plasma proteins. The performance characteristics were evaluated by gel-electrophoresis and LC-MS/MS of the bound and flow-through fractions. The capacity of a 5.6mL column was found to be 125μL of plasma. Replicate analysis demonstrated high column reproducibility and linearity, efficient removal of abundant proteins, and enrichment of lower abundance proteins observed after depletion. The novel class of affinity ligands provides an attractive alternative to traditional antibody-based immunodepletion methods.

    View details for DOI 10.1016/j.jchromb.2013.09.008

    View details for PubMedID 24090752

  • Autoantibody Signatures Involving Glycolysis and Splicesome Proteins Precede a Diagnosis of Breast Cancer among Postmenopausal Women CANCER RESEARCH Ladd, J. J., Chao, T., Johnson, M. M., Qiu, J., Chin, A., Israel, R., Pitteri, S. J., Mao, J., Wu, M., Amon, L. M., McIntosh, M., Li, C., Prentice, R., Disis, N., Hanash, S. 2013; 73 (5): 1502-1513


    We assessed the autoantibody repertoire of a mouse model engineered to develop breast cancer and the repertoire of autoantibodies in human plasmas collected at a preclinical time point and at the time of clinical diagnosis of breast cancer. In seeking to identify common pathways, networks, and protein families associated with the humoral response, we elucidated the dynamic nature of tumor antigens and autoantibody interactions. Lysate proteins from an immortalized cell line from a MMTV-neu mouse model and from MCF7 human breast cancers were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Immunoglobulin-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in tumor-bearing mice and in prediagnostic human samples. Interestingly, autoantibody reactivity was more pronounced further away than closer to diagnosis. We provide evidence for dynamic changes in autoantibody reactivity with tumor development and progression that may depend, in part, on the extent of antigen-antibody interactions.

    View details for DOI 10.1158/0008-5472.CAN-12-2560

    View details for Web of Science ID 000315741500007

    View details for PubMedID 23269276

  • Evaluation of Known Oncoantibodies, HER2, p53, and Cyclin B1, in Prediagnostic Breast Cancer Sera CANCER PREVENTION RESEARCH Lu, H., Ladd, J., Feng, Z., Wu, M., Goodell, V., Pitteri, S. J., Li, C. I., Prentice, R., Hanash, S. M., Disis, M. L. 2012; 5 (8): 1036-1043


    Serum autoantibodies, directed against oncogenic proteins, have been frequently detected in the sera of patients with breast cancer. It is unknown whether serum antibodies that are identified in patients with established disease could also be detected in patients with newly diagnosed disease or even predate the diagnosis of breast cancer. Using sera collected at the time of treatment, at the time of diagnosis, or before the time of diagnosis, the current study aimed to address the temporal relationship between breast cancer development and serum antibody response. Starting from serum antibodies to eight known breast cancer antigens, we first identified four serum antibodies, HER2/neu, p53, carcinoembryonic antigen (CEA), and cyclin B1, which are significantly increased in the sera collected from patients with breast cancer at the time of treatment. These antibodies were also elevated in breast cancer sera collected at the time of diagnosis. Finally, comparison of antibody responses in prediagnostic samples from women before the development of breast cancer and in controls showed that antibodies to the HER2/neu and p53 can be detected in sera that were collected on average more than 150 days before a breast cancer diagnosis. These results showed that serum autoantibodies commonly reported in sera from patients with established disease can also be detected in prediagnostic sera and may be useful for the early detection of breast cancer.

    View details for DOI 10.1158/1940-6207.CAPR-11-0558

    View details for Web of Science ID 000308223500006

    View details for PubMedID 22715141

  • Quantitative Proteomic Profiling Identifies Protein Correlates to EGFR Kinase Inhibition MOLECULAR CANCER THERAPEUTICS Kani, K., Faca, V. M., Hughes, L. D., Zhang, W., Fang, Q., Shahbaba, B., Luethy, R., Erde, J., Schmidt, J., Pitteri, S. J., Zhang, Q., Katz, J. E., Gross, M. E., Plevritis, S. K., McIntosh, M. W., Jain, A., Hanash, S., Agus, D. B., Mallick, P. 2012; 11 (5): 1071-1081


    Clinical oncology is hampered by lack of tools to accurately assess a patient's response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not responding to a therapy could be usefully incorporated into tools for monitoring response. Here, we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study, we use stable isotope labeling of amino acids in culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGF receptor (EGFR)-targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information, and a subset consisting of 400 proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and showed that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response.

    View details for DOI 10.1158/1535-7163.MCT-11-0852

    View details for Web of Science ID 000307984800003

    View details for PubMedID 22411897

  • Microparticles From Ovarian Carcinomas Are Shed Into Ascites and Promote Cell Migration INTERNATIONAL JOURNAL OF GYNECOLOGICAL CANCER Press, J. Z., Reyes, M., Pitteri, S. J., Pennil, C., Garcia, R., Goff, B. A., Hanash, S. M., Swisher, E. M. 2012; 22 (4): 546-552


    Microparticles are cellular-derived vesicles (0.5-1.0 μm) composed of cell membrane components, which are actively shed from the surface of various cells, including epithelial cells. We compared microparticles in ascites between women with ovarian carcinoma and women with benign ovarian pathology, and isolated tumor-derived (epithelial cell adhesion molecule [EpCAM]-positive) microparticles for functional analysis and proteomics.Cases included 8 patients with benign ovarian neoplasms and 41 with ovarian carcinoma. Ascites from a high-grade stage III serous carcinoma was used for functional and proteomic analysis. Cancer cells were isolated using EpCAM-coated beads, microparticles were isolated by ultracentrifugation/flow cytometry, and sorting was achieved using markers (eg, EpCAM). Binding and migrations assays were performed with 3 ovarian cancer cell lines. Proteomic analysis of EpCAM-positive microparticles and ascites cancer cells was performed by mass spectrometry.Microparticles in benign pelvic fluid were similar to early and advanced-stage ascites (2.4 vs 2.8 vs 2.0 × 10⁶ microparticles/mL). Advanced stage had a greater proportion of EpCAM-positive microparticles than early or benign disease (13.3% vs 2.5% vs 2.1%; P = 0.001), and serous histology had more than endometrioid (13.2% vs 1.8%; P = 0.01). Microparticles bound to the surface of 3 cultured cell lines, and were internalized into the EpCAM-positive microparticles, resulting in more cell migration than buffer alone or EpCAM-negative microparticles (P = 0.007). A dose-dependent increase was seen with increasing numbers of EpCAM-positive microparticles. Proteomics revealed that most proteins in EPCAM-positive microparticles were shared with cancer cells, and many are associated with cell motility and invasion, such as fibronectin, filamin A, vimentin, myosin-9, and fibrinogen.Ascites from advanced-stage and serous ovarian carcinomas contain large numbers of tumor-derived microparticles. In vitro, these microparticles bind to cancer cells and stimulate migration. Tumor-derived microparticles in ascites could mediate the predilection for peritoneal spread in serous ovarian carcinomas.

    View details for DOI 10.1097/IGC.0b013e318241d9b9

    View details for Web of Science ID 000303546100005

    View details for PubMedID 22315094

  • Concordant Release of Glycolysis Proteins into the Plasma Preceding a Diagnosis of ER+ Breast Cancer CANCER RESEARCH Amon, L. M., Pitteri, S. J., Li, C. I., McIntosh, M., Ladd, J. J., Disis, M., Porter, P., Wong, C. H., Zhang, Q., Lampe, P., Prentice, R. L., Hanash, S. M. 2012; 72 (8): 1935-1942


    Although the identification of peripheral blood biomarkers would enhance early detection strategies for breast cancer, the discovery of protein markers has been challenging. In this study, we sought to identify coordinated changes in plasma proteins associated with breast cancer based on large-scale quantitative mass spectrometry. We analyzed plasma samples collected up to 74 weeks before diagnosis from 420 estrogen receptor (ER)(+) cases and matched controls enrolled in the Women's Health Initiative cohort. A gene set enrichment analysis was applied to 467 quantified proteins, linking their corresponding genes to particular biologic pathways. On the basis of differences in the concentration of individual proteins, glycolysis pathway proteins exhibited a statistically significant difference between cases and controls. In particular, the enrichment was observed among cases in which blood was drawn closer to diagnosis (effect size for the 0-38 weeks prediagnostic group, 1.91; P, 8.3E-05). Analysis of plasmas collected at the time of diagnosis from an independent set of cases and controls confirmed upregulated levels of glycolysis proteins among cases relative to controls. Together, our findings indicate that the concomitant release of glycolysis proteins into the plasma is a pathophysiologic event that precedes a diagnosis of ER(+) breast cancer.

    View details for DOI 10.1158/0008-5472.CAN-11-3266

    View details for Web of Science ID 000302905700005

    View details for PubMedID 22367215

  • Increased Plasma Levels of the APC-Interacting Protein MAPRE1, LRG1, and IGFBP2 Preceding a Diagnosis of Colorectal Cancer in Women CANCER PREVENTION RESEARCH Ladd, J. J., Busald, T., Johnson, M. M., Zhang, Q., Pitteri, S. J., Wang, H., Brenner, D. E., Lampe, P. D., Kucherlapati, R., Feng, Z., Prentice, R. L., Hanash, S. M. 2012; 5 (4): 655-664


    Longitudinal blood collections from cohort studies provide the means to search for proteins associated with disease before clinical diagnosis. We investigated plasma samples from the Women's Health Initiative (WHI) cohort to determine quantitative differences in plasma proteins between subjects subsequently diagnosed with colorectal cancer (CRC) and matched controls that remained cancer-free during the period of follow-up. Proteomic analysis of WHI samples collected before diagnosis of CRC resulted in the identification of six proteins with significantly (P < 0.05) elevated concentrations in cases compared with controls. Proteomic analysis of two CRC cell lines showed that five of the six proteins were produced by cancer cells. Microtubule-associated protein RP/EB family member 1 (MAPRE1), insulin-like growth factor-binding protein 2 (IGFBP2), leucine-rich alpha-2-glycoprotein (LRG1), and carcinoembryonic antigen (CEA) were individually assayed by enzyme linked immunosorbent assay (ELISA) in 58 pairs of newly diagnosed CRC samples and controls and yielded significant elevations (P < 0.05) among cases relative to controls. A combination of these four markers resulted in a receiver operating characteristics curve with an area under the curve value of 0.841 and 57% sensitivity at 95% specificity. This combination rule was tested in an independent set of WHI samples collected within 7 months before diagnosis from cases and matched controls resulting in 41% sensitivity at 95% specificity. A panel consisting of CEA, MAPRE1, IGFBP2, and LRG1 has predictive value in prediagnostic CRC plasmas.

    View details for DOI 10.1158/1940-6207.CAPR-11-0412

    View details for Web of Science ID 000302572400020

    View details for PubMedID 22277732

  • Lung Cancer Signatures in Plasma Based on Proteome Profiling of Mouse Tumor Models CANCER CELL Taguchi, A., Politi, K., Pitteri, S. J., Lockwood, W. W., Faca, V. M., Kelly-Spratt, K., Wong, C., Zhang, Q., Chin, A., Park, K., Goodman, G., Gazdar, A. F., Sage, J., Dinulescu, D. M., Kucherlapati, R., DePinho, R. A., Kemp, C. J., Varmus, H. E., Hanash, S. M. 2011; 20 (3): 289-299


    We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models.

    View details for DOI 10.1016/j.ccr.2011.08.007

    View details for Web of Science ID 000295205700006

    View details for PubMedID 21907921

    View details for PubMedCentralID PMC3406925

  • Tumor Microenvironment-Derived Proteins Dominate the Plasma Proteome Response during Breast Cancer Induction and Progression CANCER RESEARCH Pitteri, S. J., Kelly-Spratt, K. S., Gurley, K. E., Kennedy, J., Buson, T. B., Chin, A., Wang, H., Zhang, Q., Wong, C., Chodosh, L. A., Nelson, P. S., Hanash, S. M., Kemp, C. J. 2011; 71 (15): 5090-5100


    Tumor development relies upon essential contributions from the tumor microenvironment and host immune alterations. These contributions may inform the plasma proteome in a manner that could be exploited for cancer diagnosis and prognosis. In this study, we employed a systems biology approach to characterize the plasma proteome response in the inducible HER2/neu mouse model of breast cancer during tumor induction, progression, and regression. Mass spectrometry data derived from approximately 1.6 million spectra identified protein networks involved in wound healing, microenvironment, and metabolism that coordinately changed during tumor development. The observed alterations developed prior to cancer detection, increased progressively with tumor growth and reverted toward baseline with tumor regression. Gene expression and immunohistochemical analyses suggested that the cancer-associated plasma proteome was derived from transcriptional responses in the noncancerous host tissues as well as the developing tumor. The proteomic signature was distinct from a nonspecific response to inflammation. Overall, the developing tumor simultaneously engaged a number of innate physiologic processes, including wound repair, immune response, coagulation and complement cascades, tissue remodeling, and metabolic homeostasis that were all detectable in plasma. Our findings offer an integrated view of tumor development relevant to plasma-based strategies to detect and diagnose cancer.

    View details for DOI 10.1158/0008-5472.CAN-11-0568

    View details for Web of Science ID 000293267600006

    View details for PubMedID 21653680

  • A Proteomics Platform Combining Depletion, Multi-lectin Affinity Chromatography (M-LAC), and Isoelectric Focusing to Study the Breast Cancer Proteome ANALYTICAL CHEMISTRY Zeng, Z., Hincapie, M., Pitteri, S. J., Hanash, S., Schakwijk, J., Hogan, J. M., Wang, H., Hancock, W. S. 2011; 83 (12): 4845-4854


    The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation, and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as to simultaneously detect glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation, and LC-MS analysis has been applied to discover breast cancer-associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component, and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies.

    View details for DOI 10.1021/ac2002802

    View details for Web of Science ID 000291499800035

    View details for PubMedID 21513341

    View details for PubMedCentralID PMC3148019

  • Plasma Proteome Profiles Associated with Inflammation, Angiogenesis, and Cancer PLOS ONE Kelly-Spratt, K. S., Pitteri, S. J., Gurley, K. E., Liggitt, D., Chin, A., Kennedy, J., Wong, C., Zhang, Q., Buson, T. B., Wang, H., Hanash, S. M., Kemp, C. J. 2011; 6 (5)


    Tumor development is accompanied by a complex host systemic response, which includes inflammatory and angiogenic reactions. Both tumor-derived and systemic response proteins are detected in plasma from cancer patients. However, given their non-specific nature, systemic response proteins can confound the detection or diagnosis of neoplasia. Here, we have applied an in-depth quantitative proteomic approach to analyze plasma protein changes in mouse models of subacute irritant-driven inflammation, autoreactive inflammation, and matrix associated angiogenesis and compared results to previously described findings from mouse models of polyoma middle T-driven breast cancer and Pdx1-Cre Kras(G12D) Ink4a/Arf (lox/lox)-induced pancreatic cancer. Among the confounding models, approximately 1/3 of all quantified plasma proteins exhibited a significant change in abundance compared to control mice. Of the proteins that changed in abundance, the majority were unique to each model. Altered proteins included those involved in acute phase response, inflammation, extracellular matrix remodeling, angiogenesis, and TGFβ signaling. Comparison of changes in plasma proteins between the confounder models and the two cancer models revealed proteins that were restricted to the cancer-bearing mice, reflecting the known biology of these tumors. This approach provides a basis for distinguishing between protein changes in plasma that are cancer-related and those that are part of a non-specific host response.

    View details for DOI 10.1371/journal.pone.0019721

    View details for Web of Science ID 000290531100023

    View details for PubMedID 21589862

    View details for PubMedCentralID PMC3093388

  • Lung cancer bio-signatures in plasma based on the analysis of mouse models Taguchi, A., Politi, K., Pitteri, S. J., Lockwood, W. W., Faca, V. M., Sage, J., Kemp, C. J., Varmus, H. E., Hanash, S. M. AMER ASSOC CANCER RESEARCH. 2011
  • Confounding Effects of Hormone Replacement Therapy in Protein Biomarker Studies CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Pitteri, S. J., Hanash, S. M. 2011; 20 (1): 134-139


    We have recently investigated effects of hormone replacement therapy (HRT) on the serum proteome, and found a high proportion of proteins with altered levels associated with oral estrogen and/or estrogen plus progesterone treatment. Given this finding, we have investigated the extent to which exposure to HRT may have a confounding effect in the assessment of circulating proteins as cancer biomarkers.We utilize mass spectrometry data collected from the HRT serum proteome studies to estimate the overall effect of postmenopausal hormone therapy on candidate ovarian cancer biomarkers that have been previously reported.Levels of approximately half of the proteins reported as potential ovarian cancer biomarkers were found to be affected by HRT. The impact of HRT on levels of insulin-like growth factor and inhibin protein families was found to be substantial.We conclude that the potential confounding effect of HRT and other types of exposures should be taken into consideration in cancer biomarker study design.HRT significantly affects the serum proteome and should be taken into account as part of biomarker study design and data analysis.

    View details for DOI 10.1158/1055-9965.EPI-10-0673

    View details for Web of Science ID 000285972800014

    View details for PubMedID 21037107

  • Detection of Elevated Plasma Levels of Epidermal Growth Factor Receptor Before Breast Cancer Diagnosis among Hormone Therapy Users CANCER RESEARCH Pitteri, S. J., Amon, L. M., Buson, T. B., Zhang, Y., Johnson, M. M., Chin, A., Kennedy, J., Wong, C., Zhang, Q., Wang, H., Lampe, P. D., Prentice, R. L., McIntosh, M. W., Hanash, S. M., Li, C. I. 2010; 70 (21): 8598-8606


    Applying advanced proteomic technologies to prospectively collected specimens from large studies is one means of identifying preclinical changes in plasma proteins that are potentially relevant to the early detection of diseases such as breast cancer. We conducted 14 independent quantitative proteomics experiments comparing pooled plasma samples collected from 420 estrogen receptor-positive (ER(+)) breast cancer patients ≤17 months before their diagnosis and matched controls. Based on the more than 3.4 million tandem mass spectra collected in the discovery set, 503 proteins were quantified, of which 57 differentiated cases from controls with a P value of <0.1. Seven of these proteins, for which quantitative ELISA assays were available, were assessed in an independent validation set. Of these candidates, epidermal growth factor receptor (EGFR) was validated as a predictor of breast cancer risk in an independent set of preclinical plasma samples for women overall [odds ratio (OR), 1.44; P = 0.0008] and particularly for current users of estrogen plus progestin (E + P) menopausal hormone therapy (OR, 2.49; P = 0.0001). Among current E + P users, the EGFR sensitivity for breast cancer risk was 31% with 90% specificity. Whereas the sensitivity and specificity of EGFR are insufficient for a clinically useful early detection biomarker, this study suggests that proteins that are elevated preclinically in women who go on to develop breast cancer can be discovered and validated using current proteomic technologies. Further studies are warranted to examine the role of EGFR and to discover and validate other proteins that could potentially be used for early detection of breast cancer.

    View details for DOI 10.1158/0008-5472.CAN-10-1676

    View details for Web of Science ID 000283667300038

    View details for PubMedID 20959476

  • Elafin Is a Biomarker of Graft-Versus-Host Disease of the Skin SCIENCE TRANSLATIONAL MEDICINE paczesny, s., Braun, T. M., Levine, J. E., Hogan, J., Crawford, J., Coffing, B., Olsen, S., Choi, S. W., Wang, H., Faca, V., Pitteri, S., Zhang, Q., Chin, A., Kitko, C., Mineishi, S., Yanik, G., Peres, E., Hanauer, D., Wang, Y., Reddy, P., Hanash, S., Ferrara, J. L. 2010; 2 (13)


    Graft-versus-host disease (GVHD), the major complication of allogeneic bone marrow transplantation, affects the skin, liver, and gastrointestinal tract. There are no plasma biomarkers specific for any acute GVHD target organ. We used a large-scale quantitative proteomic discovery procedure to identify biomarker candidates of skin GVHD and validated the lead candidate, elafin, with enzyme-linked immunosorbent assay in samples from 492 patients. Elafin was overexpressed in GVHD skin biopsies. Plasma concentrations of elafin were significantly higher at the onset of skin GVHD, correlated with the eventual maximum grade of GVHD, and were associated with a greater risk of death relative to other known risk factors (hazard ratio, 1.78). We conclude that elafin has significant diagnostic and prognostic value as a biomarker of skin GVHD.

    View details for DOI 10.1126/scitranslmed.3000406

    View details for Web of Science ID 000277263500004

    View details for PubMedID 20371463

  • Novel proteins associated with risk for coronary heart disease or stroke among postmenopausal women identified by in-depth plasma proteome profiling. Genome medicine Prentice, R. L., paczesny, s., Aragaki, A., Amon, L. M., Chen, L., Pitteri, S. J., McIntosh, M., Wang, P., Buson Busald, T., Hsia, J., Jackson, R. D., Rossouw, J. E., Manson, J. E., Johnson, K., Eaton, C., Hanash, S. M. 2010; 2 (7): 48-?


    Coronary heart disease (CHD) and stroke were key outcomes in the Women's Health Initiative (WHI) randomized trials of postmenopausal estrogen and estrogen plus progestin therapy. We recently reported a large number of changes in blood protein concentrations in the first year following randomization in these trials using an in-depth quantitative proteomics approach. However, even though many affected proteins are in pathways relevant to the observed clinical effects, the relationships of these proteins to CHD and stroke risk among postmenopausal women remains substantially unknown.The same in-depth proteomics platform was applied to plasma samples, obtained at enrollment in the WHI Observational Study, from 800 women who developed CHD and 800 women who developed stroke during cohort follow-up, and from 1-1 matched controls. A plasma pooling strategy, followed by extensive fractionation prior to mass spectrometry, was used to identify proteins related to disease incidence, and the overlap of these proteins with those affected by hormone therapy was examined. Replication studies, using enzyme-linked-immunosorbent assay (ELISA), were carried out in the WHI hormone therapy trial cohorts.Case versus control concentration differences were suggested for 37 proteins (nominal P < 0.05) for CHD, with three proteins, beta-2 microglobulin (B2M), alpha-1-acid glycoprotein 1 (ORM1), and insulin-like growth factor binding protein acid labile subunit (IGFALS) having a false discovery rate < 0.05. Corresponding numbers for stroke were 47 proteins with nominal P < 0.05, three of which, apolipoprotein A-II precursor (APOA2), peptidyl-prolyl isomerase A (PPIA), and insulin-like growth factor binding protein 4 (IGFBP4), have a false discovery rate < 0.05. Other proteins involved in insulin-like growth factor signaling were also highly ranked. The associations of B2M with CHD (P < 0.001) and IGFBP4 with stroke (P = 0.005) were confirmed using ELISA in replication studies, and changes in these proteins following the initiation of hormone therapy use were shown to have potential to help explain hormone therapy effects on those diseases.In-depth proteomic discovery analysis of prediagnostic plasma samples identified B2M and IGFBP4 as risk markers for CHD and stroke respectively, and provided a number of candidate markers of disease risk and candidate mediators of hormone therapy effects on CHD and identifier: NCT00000611.

    View details for DOI 10.1186/gm169

    View details for PubMedID 20667078

  • A systems approach to the proteomic identification of novel cancer biomarkers DISEASE MARKERS Pitteri, S., Hanash, S. 2010; 28 (4): 233-239


    The proteomics field has experienced rapid growth with technologies achieving ever increasing accuracy, sensitivity, and throughput, and with availability of computational tools to address particular applications. Given that the proteome represents the most functional component encoded for in the genome, a systems approach to disease investigations and biomarker identification benefits substantially from integration of proteome level studies. Here we present proteomic approaches that have allowed systematic searches for potential cancer markers by integrating cancer cell profiling with additional sources of data, as illustrated with recent studies of ovarian cancer.

    View details for DOI 10.3233/DMA-2010-0696

    View details for Web of Science ID 000279321200005

    View details for PubMedID 20534908

  • Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery PLOS ONE Pitteri, S. J., JeBailey, L., Faca, V. M., Thorpe, J. D., Silva, M. A., Ireton, R. C., Horton, M. B., Wang, H., Pruitt, L. C., Zhang, Q., Cheng, K. H., Urban, N., Hanash, S. M., Dinulescu, D. M. 2009; 4 (11)


    The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.

    View details for DOI 10.1371/journal.pone.0007916

    View details for Web of Science ID 000272004600018

    View details for PubMedID 19936259

    View details for PubMedCentralID PMC2775948

  • Application of serum proteomics to the Women's Health Initiative conjugated equine estrogens trial reveals a multitude of effects relevant to clinical findings. Genome medicine Katayama, H., paczesny, s., Prentice, R., Aragaki, A., Faca, V. M., Pitteri, S. J., Zhang, Q., Wang, H., Silva, M., Kennedy, J., Rossouw, J., Jackson, R., Hsia, J., Chlebowski, R., Manson, J., Hanash, S. 2009; 1 (4): 47-?


    The availability of serum collections from the Women's Health Initiative (WHI) conjugated equine estrogens (CEE) randomized controlled trial provides an opportunity to test the potential of in-depth quantitative proteomics to uncover changes in the serum proteome related to CEE and to assess their relevance to trial findings, including elevations in the risk of stroke and venous thromboembolism and a reduction in fractures.Five independent large scale quantitative proteomics analyses were performed, each comparing a set of pooled serum samples collected from 10 subjects, 1 year following initiation of CEE at 0.625 mg/d, relative to their baseline pool. A subset of proteins that exhibited increased levels with CEE by quantitative proteomics was selected for validation studies.Of 611 proteins quantified based on differential stable isotope labeling, the levels of 116 (19%) were changed after 1 year of CEE (nominal P < 0.05), while 64 of these had estimated false discovery rates <0.05. Most of the changed proteins were not previously known to be affected by CEE and had relevance to processes that included coagulation, metabolism, osteogenesis, inflammation, and blood pressure maintenance. To validate quantitative proteomic data, 14 proteins were selected for ELISA. Findings for ten - IGF1, IGFBP4, IGFBP1, IGFBP2, F10, AHSG, GC, CP, MMP2, and PROZ - were confirmed in the initial set of 50 subjects and further validated in an independent set of 50 additional subjects who received CEE.CEE affected a substantial fraction of the serum proteome, including proteins with relevance to findings from the WHI CEE trial related to cardiovascular disease and identifier: NCT00000611.

    View details for DOI 10.1186/gm47

    View details for PubMedID 19402886

  • Postmenopausal estrogen and progestin effects on the serum proteome. Genome medicine Pitteri, S. J., Hanash, S. M., Aragaki, A., Amon, L. M., Chen, L., Busald Buson, T., paczesny, s., Katayama, H., Wang, H., Johnson, M. M., Zhang, Q., McIntosh, M., Wang, P., Kooperberg, C., Rossouw, J. E., Jackson, R. D., Manson, J. E., Hsia, J., Liu, S., Martin, L., Prentice, R. L. 2009; 1 (12): 121-?


    Women's Health Initiative randomized trials of postmenopausal hormone therapy reported intervention effects on several clinical outcomes, with some important differences between estrogen alone and estrogen plus progestin. The biologic mechanisms underlying these effects, and these differences, have yet to be fully elucidated.Baseline serum samples were compared with samples drawn 1 year later for 50 women assigned to active hormone therapy in both the estrogen-plus-progestin and estrogen-alone randomized trials, by applying an in-depth proteomic discovery platform to serum pools from 10 women per pool.In total, 378 proteins were quantified in two or more of the 10 pooled serum comparisons, by using strict identification criteria. Of these, 169 (44.7%) showed evidence (nominal P < 0.05) of change in concentration between baseline and 1 year for one or both of estrogen-plus-progestin and estrogen-alone groups. Quantitative changes were highly correlated between the two hormone-therapy preparations. A total of 98 proteins had false discovery rates < 0.05 for change with estrogen plus progestin, compared with 94 for estrogen alone. Of these, 84 had false discovery rates <0.05 for both preparations. The observed changes included multiple proteins relevant to coagulation, inflammation, immune response, metabolism, cell adhesion, growth factors, and osteogenesis. Evidence of differential changes also was noted between the hormone preparations, with the strongest evidence in growth factor and inflammation pathways.Serum proteomic analyses yielded a large number of proteins similarly affected by estrogen plus progestin and by estrogen alone and identified some proteins and pathways that appear to be differentially affected between the two hormone preparations; this may explain their distinct clinical effects.

    View details for DOI 10.1186/gm121

    View details for PubMedID 20034393

  • Occurrence of Autoantibodies to Annexin I, 14-3-3 Theta and LAMR1 in Prediagnostic Lung Cancer Sera JOURNAL OF CLINICAL ONCOLOGY Qiu, J., Choi, G., Li, L., Wang, H., Pitteri, S. J., Pereira-Faca, S. R., Krasnoselsky, A. L., Randolph, T. W., Omenn, G. S., Edelstein, C., Barnett, M. J., Thornquist, M. D., Goodman, G. E., Brenner, D. E., Feng, Z., Hanash, S. M. 2008; 26 (31): 5060-5066


    We have implemented a high throughput platform for quantitative analysis of serum autoantibodies, which we have applied to lung cancer for discovery of novel antigens and for validation in prediagnostic sera of autoantibodies to antigens previously defined based on analysis of sera collected at the time of diagnosis.Proteins from human lung adenocarcinoma cell line A549 lysates were subjected to extensive fractionation. The resulting 1,824 fractions were spotted in duplicate on nitrocellulose-coated slides. The microarrays produced were used in a blinded validation study to determine whether annexin I, PGP9.5, and 14-3-3 theta antigens previously found to be targets of autoantibodies in newly diagnosed patients with lung cancer are associated with autoantibodies in sera collected at the presymptomatic stage and to determine whether additional antigens may be identified in prediagnostic sera. Individual sera collected from 85 patients within 1 year before a diagnosis of lung cancer and 85 matched controls from the Carotene and Retinol Efficacy Trial (CARET) cohort were hybridized to individual microarrays.We present evidence for the occurrence in lung cancer sera of autoantibodies to annexin I, 14-3-3 theta, and a novel lung cancer antigen, LAMR1, which precede onset of symptoms and diagnosis.Our findings suggest potential utility of an approach to diagnosis of lung cancer before onset of symptoms that includes screening for autoantibodies to defined antigens.

    View details for DOI 10.1200/JCO.2008.16.2388

    View details for Web of Science ID 000260537600012

    View details for PubMedID 18794547

    View details for PubMedCentralID PMC2652098

  • Precursor-ion mass re-estimation improves peptide identification on hybrid instruments JOURNAL OF PROTEOME RESEARCH Luethy, R., Kessner, D. E., Katz, J. E., McLean, B., Grothe, R., Kani, K., Faca, V., Pitteri, S., Hanash, S., Agus, D. B., Mallick, P. 2008; 7 (9): 4031-4039


    Mass spectrometry-based proteomics experiments have become an important tool for studying biological systems. Identifying the proteins in complex mixtures by assigning peptide fragmentation spectra to peptide sequences is an important step in the proteomics process. The 1-2 ppm mass-accuracy of hybrid instruments, like the LTQ-FT, has been cited as a key factor in their ability to identify a larger number of peptides with greater confidence than competing instruments. However, in replicate experiments of an 18-protein mixture, we note parent masses deviate 171 ppm, on average, for ion-trap data directed identifications and 8 ppm, on average, for preview Fourier transform (FT) data directed identifications. These deviations are neither caused by poor calibration nor by excessive ion-loading and are most likely due to errors in parent mass estimation. To improve these deviations, we introduce msPrefix, a program to re-estimate a peptide's parent mass from an associated high-accuracy full-scan survey spectrum. In 18-protein mixture experiments, msPrefix parent mass estimates deviate only 1 ppm, on average, from the identified peptides. In a cell lysate experiment searched with a tolerance of 50 ppm, 2295 peptides were confidently identified using native data and 4560 using msPrefixed data. Likewise, in a plasma experiment searched with a tolerance of 50 ppm, 326 peptides were identified using native data and 1216 using msPrefixed data. msPrefix is also able to determine which MS/MS spectra were possibly derived from multiple precursor ions. In complex mixture experiments, we demonstrate that more than 50% of triggered MS/MS may have had multiple precursor ions and note that spectra with multiple candidate ions are less likely to result in an identification using TANDEM. These results demonstrate integration of msPrefix into traditional shotgun proteomics workflows significantly improves identification results.

    View details for DOI 10.1021/pr800307m

    View details for Web of Science ID 000259015500038

    View details for PubMedID 18707148

  • Proteomic Analysis of Ovarian Cancer Cells Reveals Dynamic Processes of Protein Secretion and Shedding of Extra-Cellular Domains PLOS ONE Faca, V. M., Ventura, A. P., Fitzgibbon, M. P., Pereira-Faca, S. R., Pitteri, S. J., Green, A. E., Ireton, R. C., Zhang, Q., Wang, H., O'Briant, K. C., Drescher, C. W., Schummer, M., McIntosh, M. W., Knudsen, B. S., Hanash, S. M. 2008; 3 (6)


    Elucidation of the repertoire of secreted and cell surface proteins of tumor cells is relevant to molecular diagnostics, tumor imaging and targeted therapies. We have characterized the cell surface proteome and the proteins released into the extra-cellular milieu of three ovarian cancer cell lines, CaOV3, OVCAR3 and ES2 and of ovarian tumor cells enriched from ascites fluid.To differentiate proteins released into the media from protein constituents of media utilized for culture, cells were grown in the presence of [(13)C]-labeled lysine. A biotinylation-based approach was used to capture cell surface associated proteins. Our general experimental strategy consisted of fractionation of proteins from individual compartments followed by proteolytic digestion and LC-MS/MS analysis. In total, some 6,400 proteins were identified with high confidence across all specimens and fractions.Protein profiles of the cell lines had substantial similarity to the profiles of human ovarian cancer cells from ascites fluid and included protein markers known to be associated with ovarian cancer. Proteomic analysis indicated extensive shedding from extra-cellular domains of proteins expressed on the cell surface, and remarkably high secretion rates for some proteins (nanograms per million cells per hour). Cell surface and secreted proteins identified by in-depth proteomic profiling of ovarian cancer cells may provide new targets for diagnosis and therapy.

    View details for DOI 10.1371/journal.pone.0002425

    View details for Web of Science ID 000263280700011

    View details for PubMedID 18560578

    View details for PubMedCentralID PMC2409963

  • Isoform analysis of LC-MS/MS data from multidimensional fractionation of the serum proteome JOURNAL OF PROTEOME RESEARCH Krasnoselsky, A. L., Faca, V. M., Pitteri, S. J., Zhang, Q., Hanash, S. M. 2008; 7 (6): 2546-2552


    We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states.

    View details for DOI 10.1021/pr7007219

    View details for Web of Science ID 000256599000037

    View details for PubMedID 18419151

  • A mouse to human search for plasma Proteome changes associated with pancreatic tumor development PLOS MEDICINE Faca, V. M., Song, K. S., Wang, H., Zhang, Q., Krasnoselsky, A. L., Newcomb, L. F., Plentz, R. R., Gurumurthy, S., Redston, M. S., Pitteri, S. J., Pereira-Faca, S. R., Ireton, R. C., Katayama, H., Glukhova, V., Phanstiel, D., Brenner, D. E., Anderson, M. A., Misek, D., Scholler, N., Urban, N. D., Barnett, M. J., Edelstein, C., Goodman, G. E., Thornquist, M. D., McIntosh, M. W., DePinho, R. A., Bardeesy, N., Hanash, S. M. 2008; 5 (6): 953-967


    The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer.Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer.Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection.

    View details for DOI 10.1371/journal.pmed.0050123

    View details for Web of Science ID 000257105600020

    View details for PubMedID 18547137

  • Mining the plasma proteome for cancer biomarkers NATURE Hanash, S. M., Pitteri, S. J., Faca, V. M. 2008; 452 (7187): 571-579


    Systematic searches for plasma proteins that are biological indicators, or biomarkers, for cancer are underway. The difficulties caused by the complexity of biological-fluid proteomes and tissue proteomes (which contribute proteins to plasma) and by the extensive heterogeneity among diseases, subjects and levels of sample procurement are gradually being overcome. This is being achieved through rigorous experimental design and in-depth quantitative studies. The expected outcome is the development of panels of biomarkers that will allow early detection of cancer and prediction of the probable response to therapy. Achieving these objectives requires high-quality specimens with well-matched controls, reagent resources, and an efficient process to confirm discoveries through independent validation studies.

    View details for DOI 10.1038/nature06916

    View details for Web of Science ID 000254567200035

    View details for PubMedID 18385731

  • Plasma proteome profiling of a mouse model of breast cancer identifies a set of up-regulated proteins in common with human breast cancer cells JOURNAL OF PROTEOME RESEARCH Pitteri, S. J., Faca, V. M., Kelly-Spratt, K. S., Kasarda, A. E., Wang, H., Zhang, Q., Newcomb, L., Krasnoselsky, A., paczesny, s., Choi, G., Fitzgibbon, M., McIntosh, M. W., Kemp, C. J., Hanash, S. M. 2008; 7 (4): 1481-1489


    We have applied an in-depth quantitative proteomic approach, combining isotopic labeling extensive intact protein separation and mass spectrometry, for high confidence identification of protein changes in plasmas from a mouse model of breast cancer. We hypothesized that a wide spectrum of proteins may be up-regulated in plasma with tumor development and that comparisons with proteins expressed in human breast cancer cell lines may identify a subset of up-regulated proteins in common with proteins expressed in breast cancer cell lines that may represent candidate biomarkers for breast cancer. Plasma from PyMT transgenic tumor-bearing mice and matched controls were obtained at two time points during tumor growth. A total of 133 proteins were found to be increased by 1.5-fold or greater at one or both time points. A comparison of this set of proteins with published findings from proteomic analysis of human breast cancer cell lines yielded 49 proteins with increased levels in mouse plasma that were identified in breast cancer cell lines. Pathway analysis comparing the subset of up-regulated proteins known to be expressed in breast cancer cell lines with other up-regulated proteins indicated a cancer related function for the former and a host-response function for the latter. We conclude that integration of proteomic findings from mouse models of breast cancer and from human breast cancer cell lines may help identify a subset of proteins released by breast cancer cells into the circulation and that occur at increased levels in breast cancer.

    View details for DOI 10.1021/pr7007994

    View details for Web of Science ID 000254711000013

    View details for PubMedID 18311905

  • A mouse plasma peptide atlas as a resource for disease proteomics GENOME BIOLOGY Zhang, Q., Menon, R., Deutsch, E. W., Pitteri, S. J., Faca, V. M., Wang, H., Newcomb, L. F., DePinho, R. A., Bardeesy, N., Dinulescu, D., Hung, K. E., Kucherlapati, R., Jacks, T., Politi, K., Aebersold, R., Omenn, G. S., States, D. J., Hanash, S. M. 2008; 9 (6)


    We present an in-depth analysis of mouse plasma leading to the development of a publicly available repository composed of 568 liquid chromatography-tandem mass spectrometry runs. A total of 13,779 distinct peptides have been identified with high confidence. The corresponding approximately 3,000 proteins are estimated to span a 7 logarithmic range of abundance in plasma. A major finding from this study is the identification of novel isoforms and transcript variants not previously predicted from genome analysis.

    View details for DOI 10.1186/gb-2008-9-6-r93

    View details for Web of Science ID 000257498000012

    View details for PubMedID 18522751

    View details for PubMedCentralID PMC2481425

  • Proteomic approaches for cancer biomarker discovery in plasma EXPERT REVIEW OF PROTEOMICS Pitteri, S. J., Hanash, S. M. 2007; 4 (5): 589-590

    View details for DOI 10.1586/14789450.4.5.589

    View details for Web of Science ID 000250658000001

    View details for PubMedID 17941811

  • Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes JOURNAL OF PROTEOME RESEARCH Faca, V., Pitteri, S. J., Newcomb, L., Glukhova, V., Phanstiel, D., Krasnoselsky, A., Zhang, Q., Struthers, J., Wang, H., Eng, J., Fitzgibbon, M., McIntosh, M., Hanash, S. 2007; 6 (9): 3558-3565


    In-depth analysis of the serum and plasma proteomes by mass spectrometry is challenged by the vast dynamic range of protein abundance and substantial complexity. There is merit in reducing complexity through fractionation to facilitate mass spectrometry analysis of low-abundance proteins. However, fractionation reduces throughput and has the potential of diluting individual proteins or inducing their loss. Here, we have investigated the contribution of extensive fractionation of intact proteins to depth of analysis. Pooled serum depleted of abundant proteins was fractionated by an orthogonal two-dimensional system consisting of anion-exchange and reversed-phase chromatography. The resulting protein fractions were aliquotted; one aliquot was analyzed by shotgun LC-MS/MS, and another was further resolved into protein bands in a third dimension using SDS-PAGE. Individual gel bands were excised and subjected to in situ digestion and mass spectrometry. We demonstrate that increased fractionation results in increased depth of analysis based on total number of proteins identified in serum and based on representation in individual fractions of specific proteins identified in gel bands following a third-dimension SDS gel analysis. An intact protein analysis system (IPAS) based on a two-dimensional plasma fractionation schema was implemented that resulted in identification of 1662 proteins with high confidence with representation of protein isoforms that differed in their chromatographic mobility. Further increase in depth of analysis was accomplished by repeat analysis of aliquots from the same set of two-dimensional fractions resulting in overall identification of 2254 proteins. We conclude that substantial depth of analysis of proteins from milliliter quantities of serum or plasma and detection of isoforms are achieved with depletion of abundant proteins followed by two-dimensional protein fractionation and MS analysis of individual fractions.

    View details for DOI 10.1021/pr070233q

    View details for Web of Science ID 000249371000023

    View details for PubMedID 17696519

  • Ion/molecule reactions of cation radicals formed from protonated polypeptides via gas-phase ion/ion electron transfer JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Xia, Y., Chrisman, P. A., Pitteri, S. J., Erickson, D. E., McLuckey, S. A. 2006; 128 (36): 11792-11798


    Cation radicals formed via gas-phase electron transfer to multiply protonated polypeptides have been found to react with molecular oxygen. Such cation radicals are of interest within the context of electron transfer dissociation, a phenomenon with high utility for the characterization of peptide and protein primary structures. Most of the cation radicals show the attachment of O(2) under room temperature storage conditions in an electrodynamic ion trap. At higher temperatures and under conditions of collisional activation, the oxygen adduct species lose O(2), HO(*), or HO(2)(*), depending upon the identity of the side chain at the radical site. The fragments containing the C-terminus, the so-called z-ions, which are predominantly radical species, engage in reactions with molecular oxygen. This allows for the facile distinction between z-ions and their complementary even-electron c-ion counterparts. Such a capability has utility in protein identification and characterization via mass spectrometry. Intact electron transfer products also show oxygen attachment. Subsequent activation of such adducts show dissociation behavior very similar to that noted for z-ion adducts. These observations indicate that ion/radical reactions can be used to probe the locations of radical sites in the undissociated electron transfer products as well as distinguish between c- and z-type ions.

    View details for DOI 10.1021/ja063248i

    View details for Web of Science ID 000240291900027

    View details for PubMedID 16953618

  • Charge-state dependent dissociation of a trypsin/inhibitor complex via ion trap collisional activation INTERNATIONAL JOURNAL OF MASS SPECTROMETRY Pitteri, S. J., Chrisman, P. A., Badman, E. R., McLuckey, S. A. 2006; 253 (3): 147-155
  • Parallel ion parking of protein mixtures ANALYTICAL CHEMISTRY Chrisman, P. A., Pitteri, S. J., McLuckey, S. A. 2006; 78 (1): 310-316


    The multiple charging phenomenon resulting from electrospray ionization of proteins, while useful for the ability to make several mass measurements on a single component, can lead to highly complex spectra when mixtures are analyzed, as each component can generate multiple ions of distinct mass-to-charge ratio. Ion/ion proton-transfer reactions can overcome this problem by reduction of all components to the +1 charge state, but this typically requires the ability to extend the mass range of the instrument well beyond that available in most commercial instruments. Furthermore, reduction of protein charge to +1 also results in a reduction in detector response. Here it is shown that application of a relatively high amplitude, low-frequency auxiliary ac signal to the end cap electrodes of a 3-D ion trap during an ion/ion reaction can slow the ion/ion reaction rates of ions over a broad m/z range, in a process termed HALF parallel ion parking. Adjustment of the frequency and amplitude of the applied voltage allows the mass range into which the initial ion signal is moved to be controlled, allowing for the simplification of multicomponent mixtures within a mass range that is more commonly available on commercial systems. In addition to decreasing spectral complexity, this is advantageous for mixtures with low-abundance components, as there is less compromise with detector response than in reduction to the +1 charge state. Preliminary evidence also suggests that the ion collision cross section may play an important role in determining which charge states are most significantly inhibited from further ion/ion reactions under a given set of ion parking conditions.

    View details for DOI 10.1021/ac0515778

    View details for Web of Science ID 000234312300047

    View details for PubMedID 16383342

  • Differentiation of aspartic and isoaspartic acids using electron transfer dissociation JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY O'Connor, P. B., Cournoyer, J. J., Pitteri, S. J., Chrisman, P. A., McLuckey, S. A. 2006; 17 (1): 15-19


    Electron-transfer dissociation allows differentiation of isoaspartic acid and aspartic acid residues using the same c + 57 and z - 57 peaks that were previously observed with electron capture dissociation. These peaks clearly define both the presence and the position of isoaspartic acid residues and they are relatively abundant. The lower resolution of the ion trap instrument makes detection of the aspartic acid residue's diagnostic peak difficult because of interference with side-chain fragment ions from arginine residues, but the aspartic acid residues are still clearly observed in the backbone cleavages and can be inferred from the absence of the isoaspartic acid diagnostic ions.

    View details for DOI 10.1016/j.jasms.2005.08.019

    View details for Web of Science ID 000234520000003

    View details for PubMedID 16338146

  • Recent developments in the ion/ion chemistry of high-mass multiply charged ions MASS SPECTROMETRY REVIEWS Pitteri, S. J., McLuckey, S. A. 2005; 24 (6): 931-958


    The ability to form multiply charged high-mass ions in the gas-phase, most notably via electrospray ionization (ESI), has allowed the study of many different combinations of positively and negatively charged ions. The charged products are directly amenable to study with mass spectrometry. Ion/ion reactions have proved to be "universal" in the sense that the high exothermicities and large rate constants associated with essentially any combination of oppositely charged ions lead to reaction regardless of the chemical functionalities associated with the ions. These characteristics make ion/ion reactions potentially analytically useful provided reagent ion densities and spatial overlap of the oppositely charged ions are high. These conditions can be readily met by several instrumental configurations. The focus of this review is to highlight developments in this field since 1998. Novel instrumentation has been developed to study ion/ion reactions, such as atmospheric pressure ion/ion reactors followed by mass analysis, or electrodynamic ion trap mass spectrometers, which are used as reaction vessels at sub-atmospheric pressures. A wide variety of reaction phenomenologies have been observed in various ion/ion reactions, with proton transfer being the most common. New phenomenologies have been observed in the reactions of multiply charged positive ions with singly charged negative ions, including cation transfer and cation exchange. A new series of reactions between multiply charged positive ions and multiply charged negative ions have been made possible by recent instrumentation developments. These reactions have led to the observation of proton transfer and complex formation. These observations have provided new insights into ion/ion reaction dynamics and a bound orbit model appears to best account for experimental results. New applications are also discussed for a several ion/ion reaction.

    View details for DOI 10.1002/mas.20048

    View details for Web of Science ID 000232751800010

    View details for PubMedID 15706594

  • Electron transfer versus proton transfer in gas-phase ion/ion reactions of polyprotonated peptides JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Gunawardena, H. P., He, M., Chrisman, P. A., Pitteri, S. J., Hogan, J. M., Hodges, B. D., McLuckey, S. A. 2005; 127 (36): 12627-12639


    The ion/ion reactions of several dozen reagent anions with triply protonated cations of the model peptide KGAILKGAILR have been examined to evaluate predictions of a Landau-Zener-based model for the likelihood for electron transfer. Evidence for electron transfer was provided by the appearance of fragment ions unique to electron transfer or electron capture dissociation. Proton transfer and electron transfer are competitive processes for any combination of anionic and cationic reactants. For reagent anions in reactions with protonated peptides, proton transfer is usually significantly more exothermic than electron transfer. If charge transfer occurs at relatively long distances, electron transfer should, therefore, be favored on kinetic grounds because the reactant and product channels cross at greater distances, provided conditions are favorable for electron transfer at the crossing point. The results are consistent with a model based on Landau-Zener theory that indicates both thermodynamic and geometric criteria apply for electron transfer involving polyatomic anions. Both the model and the data suggest that electron affinities associated with the anionic reagents greater than about 60-70 kcal/mol minimize the likelihood that electron transfer will be observed. Provided the electron affinity is not too high, the Franck-Condon factors associated with the anion and its corresponding neutral must not be too low. When one or the other of these criteria is not met, proton transfer tends to occur essentially exclusively. Experiments involving ion/ion attachment products also suggest that a significant barrier exists to the isomerization between chemical complexes that, if formed, lead to either proton transfer or electron transfer.

    View details for DOI 10.1021/ja0526057

    View details for Web of Science ID 000232039100053

    View details for PubMedID 16144411

    View details for PubMedCentralID PMC1570753

  • Electron-transfer ion/ion reactions of doubly protonated peptides: Effect of elevated bath gas temperature ANALYTICAL CHEMISTRY Pitteri, S. J., Chrisman, P. A., McLuckey, S. A. 2005; 77 (17): 5662-5669


    In this study, the electron-transfer dissociation (ETD) behavior of cations derived from 27 different peptides (22 of which are tryptic peptides) has been studied in a 3D quadrupole ion trap mass spectrometer. Ion/ion reactions between peptide cations and nitrobenzene anions have been examined at both room temperature and in an elevated temperature bath gas environment to form ETD product ions. From the peptides studied, the ETD sequence coverage tends to be inversely related to peptide size. At room temperature, very high sequence coverage (approximately 100%) was observed for small peptides (< or =7 amino acids). For medium-sized peptides composed of 8-11 amino acids, the average sequence coverage was 46%. Larger peptides with 14 or more amino acids yielded an average sequence coverage of 23%. Elevated-temperature ETD provided increased sequence coverage over room-temperature experiments for the peptides of greater than 7 residues, giving an average of 67% for medium-sized peptides and 63% for larger peptides. Percent ETD, a measure of the extent of electron transfer, has also been calculated for the peptides and also shows an inverse relation with peptide size. Bath gas temperature does not have a consistent effect on percent ETD, however. For the tryptic peptides, fragmentation is localized at the ends of the peptides suggesting that the distribution of charge within the peptide may play an important role in determining fragmentation sites. A triply protonated peptide has also been studied and shows behavior similar to the doubly charged peptides. These preliminary results suggest that for a given charge state there is a maximum size for which high sequence coverage is obtained and that increasing the bath gas temperature can increase this maximum.

    View details for DOI 10.1021/ac050666h

    View details for Web of Science ID 000231652300043

    View details for PubMedID 16131079

    View details for PubMedCentralID PMC1356655

  • SO2- electron transfer ion/ion reactions with disulfide linked polypeptide ions JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Chrisman, P. A., Pitteri, S. J., Hogan, J. M., McLuckey, S. A. 2005; 16 (7): 1020-1030


    Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO2-* in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)-S* and Chain A (or B)-SH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M + 2H]+* ions, which appear to be less kinetically stable than the proton transfer [M + H]+ product. When subjected to collision-induced dissociation (CID), the [M + 2H]+* ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M + 2H]+* ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each.

    View details for DOI 10.1016/j.jasms.2005.02.010

    View details for Web of Science ID 000230045500006

    View details for PubMedID 15914021

    View details for PubMedCentralID PMC1356657

  • Parallel ion parking: Improving conversion of parents to first-generation products in electron transfer dissociation ANALYTICAL CHEMISTRY Chrisman, P. A., Pitteri, S. J., McLuckey, S. A. 2005; 77 (10): 3411-3414


    Electron-transfer dissociation (ETD) in a tandem mass spectrometer is an analytically useful ion/ion reaction technique for deriving polypeptide sequence information, but its utility can be limited by sequential reactions of the products. Sequential reactions lead to neutralization of some products, as well as to signals from products derived from multiple cleavages that can be difficult to interpret. A method of inhibiting sequential ETD fragmentation in a quadrupole ion trap is demonstrated here for the reaction of a triply protonated peptide with nitrobenzene anions. A tailored waveform (in this case, a filtered noise field) is applied during the ion/ion reaction time to accelerate simultaneously first-generation product ions and thereby inhibit their further reaction. This results in a approximately 50% gain in the relative yield of first-generation products and allows for the conversion of more than 90% of the original parent ions into first-generation products. Gains are expected to be even larger when higher charge-state cations are used, as the rates of sequential reaction become closer to the initial reaction rate.

    View details for DOI 10.1021/ac0503613

    View details for Web of Science ID 000229206800050

    View details for PubMedID 15889938

    View details for PubMedCentralID PMC1350601

  • Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: Reactions of doubly and triply protonated peptides with SO2 center dot- ANALYTICAL CHEMISTRY Pitteri, S. J., Chrisman, P. A., Hogan, J. M., McLuckey, S. A. 2005; 77 (6): 1831-1839


    Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.

    View details for DOI 10.1021/ac0483872

    View details for Web of Science ID 000227759500039

    View details for PubMedID 15762593

  • Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation JOURNAL OF PROTEOME RESEARCH Hogan, J. M., Pitteri, S. J., Chrisman, P. A., McLuckey, S. A. 2005; 4 (2): 628-632


    Glycosylation is an important post-translational modification. Analysis of glycopeptides is difficult using collision-induced dissociation, as it typically yields only information about the glycan structure, without any peptide sequence information. We demonstrate here how a 3D-quadrupole ion trap, using the complementary techniques of collision induced dissociation (CID) and electron-transfer dissociation (ETD), can be used to elucidate the glycan structure and peptide sequence of the N-glycosylated peptide from a fractionated tryptic digest of the lectin from the coral tree, Erythina cristagalli. CID experiments on the multiply protonated glycopeptide ions yield, almost exclusively, cleavage at glycosidic bonds, with little peptide backbone fragmentation. ETD reactions of the triply charged glycopeptide cations with either sulfur dioxide or nitrobenzene anions yield cleavage of the peptide backbone with no loss of the glycan structure. These results show that a 3D-quadrupole ion trap can be used to provide glycopeptide amino acid sequence information as well as information about the glycan structure.

    View details for DOI 10.1021/pr049770q

    View details for Web of Science ID 000228421900051

    View details for PubMedID 15822944

    View details for PubMedCentralID PMC1350609

  • Effects of single amino acid substitution on the collision-induced dissociation of intact protein ions: Turkey ovomucoid third domain JOURNAL OF PROTEOME RESEARCH Newton, K. A., Pitteri, S. J., Laskowski, M., McLuckey, S. A. 2004; 3 (5): 1033-1041


    Expanded understanding of the factors that direct polypeptide ion fragmentation can lead to improved specificity in the use of tandem mass spectrometry for the identification and characterization of proteins. Like the fragmentation of peptide cations, the dissociation of whole protein cations shows several preferred cleavages, the likelihood for which is parent ion charge dependent. While such cleavages are often observed, they are far from universally observed, despite the presence of the residues known to promote them. Furthermore, cleavages at residues not noted to be common in a variety of proteins can be dominant for a particular protein or protein ion charge state. Motivated by the ability to study a small protein, turkey ovomucoid third domain, for which a variety of single amino acid variants are available, the effects of changing the identity of one amino acid in the protein sequence on its dissociation behavior were examined. In particular, changes in amino acids associated with C-terminal aspartic acid cleavage and N-terminal proline cleavage were emphasized. Consistent with previous studies, the product ion spectra were found to be dependent upon the parent ion charge state. Furthermore, the fraction of possible C-terminal aspartic acid cleavages observed to occur for this protein was significantly larger than the fraction of possible N-terminal proline cleavages. In fact, very little N-terminal proline cleavage was noted for the wild-type protein despite the presence of three proline residues in the protein. The addition/removal of proline and aspartic acids was studied along with changes in selected residues adjacent to proline residues. Evidence for inhibition of proline cleavage by the presence of nearby basic residues was noted, particularly if the basic residue was likely to be protonated.

    View details for DOI 10.1021/pr049910w

    View details for Web of Science ID 000224693800015

    View details for PubMedID 15473693

    View details for PubMedCentralID PMC1350662

  • Affecting proton mobility in activated peptide and whole protein ions via lysine guanidination JOURNAL OF PROTEOME RESEARCH Pitteri, S. J., Reid, G. E., McLuckey, S. A. 2004; 3 (1): 46-54


    We have evaluated the effect of lysine guanidination in peptides and proteins on the dissociation of protonated ions in the gas phase. The dissociation of guanidinated model peptide ions compared to their unmodified forms showed behavior consistent with concepts of proton mobility as a major factor in determining favored fragmentation channels. Reduction of proton mobility associated with lysine guanidination was reflected by a relative increase in cleavages occurring C-terminal to aspartic acid residues as well as increases in small molecule losses. To evaluate the effect of guanidination on the dissociation behavior of whole protein ions, bovine ubiquitin was selected as a model. Essentially, all of the amide bond cleavages associated with the +10 charge state of fully guanidinated ubiquitin were observed to occur C-terminal to aspartic acid residues, unlike the dissociation behavior of the +10 ion of the unmodified protein, where competing cleavage N-terminal to proline and nonspecific amide bond cleavages were also observed. The +8 and lower charge states of the guanidinated protein showed prominent losses of small neutral molecules. This overall fragmentation behavior is consistent with current hypotheses regarding whole protein dissociation that consider proton mobility and intramolecular charge solvation as important factors in determining favored dissociation channels, and are also consistent with the fragmentation behaviors observed for the guanidinated model peptide ions. Further evaluation of the utility of condensed phase guanidination of whole proteins is necessary but the results described here confirm that guanidination can be an effective strategy for enhancing C-terminal aspartic acid cleavages. Gas phase dissociation exclusively at aspartic acid residues, especially for whole protein ions, could be useful in identifying and characterizing proteins via tandem mass spectrometry of whole protein ions.

    View details for DOI 10.1021/pr034054u

    View details for Web of Science ID 000189022500004

    View details for PubMedID 14998162

  • Phosphorylation site identification via ion trap tandem mass spectrometry of whole protein and peptide ions: Bovine alpha-crystallin A chain ANALYTICAL CHEMISTRY Hogan, J. M., Pitteri, S. J., McLuckey, S. A. 2003; 75 (23): 6509-6516


    Tandem mass spectrometry was applied both to ions of a tryptic fragment and intact protein of bovine alpha-crystallin A chain to localize the single site of phosphorylation. The [M + 19H](19+) to [M + 11H](11+) charge states of both phosphorylated and unphosphorylated bovine alpha-crystallin A chain whole protein ions were subjected to collisional activation in a quadrupole ion trap. Ion parking was used to increase the number of parent ions over that yielded by electrospray. Ion-ion proton-transfer reactions were used to reduce the product ion charge states largely to +1 to simplify spectral interpretation. In agreement with previous studies on whole protein ion fragmentation, both protein forms showed backbone cleavages C-terminal to aspartic acid residues at lower charge states. The phosphorylated protein showed competitive fragmentation between backbone cleavage and the neutral loss of phosphoric acid. Analysis of which backbone cleavage products did or did not contain the phosphate was used to localize the site of phosphorylation to one of two possible serine residues. A tryptic digest of the bovine alpha-crystallin A chain yielded a phosphopeptide containing one missed cleavage site. The peptide provided information complementary to that obtained from the intact protein and localized the modified serine to residue 122. Fragmentation of the triply charged phosphopeptide yielded five possible serine phosphorylation sites. Fragmentation of the doubly charged phosphopeptide, formed by ion/ion proton-transfer reactions, positively identified the phosphorylation site as serine-122.

    View details for DOI 10.1021/ac034410s

    View details for Web of Science ID 000186986000023

    View details for PubMedID 14640721

  • A Chemical Ionization Technique for Measurement of Pernitric Acid in the Upper Troposphere Geophys Res Lett Slusher DL, Pitteri SJ, Haman BJ, Tanner DJ, Huey LG 2001; 28 (20): 3875-78
  • Osmium(IV) complexes TpOs(X)Cl-2 and their Os(III) counterparts: oxidizing compounds with an unusual resistance to ligand substitution JOURNAL OF THE CHEMICAL SOCIETY-DALTON TRANSACTIONS Bennett, B. K., Pitteri, S. J., Pilobello, L., Lovell, S., Kaminsky, W., Mayer, J. M. 2001: 3489-3497

    View details for DOI 10.1039/b103993h

    View details for Web of Science ID 000172669900017